WO2017222330A1 - Recombinant single-chain fviii and chemical conjugate thereof - Google Patents

Recombinant single-chain fviii and chemical conjugate thereof Download PDF

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WO2017222330A1
WO2017222330A1 PCT/KR2017/006633 KR2017006633W WO2017222330A1 WO 2017222330 A1 WO2017222330 A1 WO 2017222330A1 KR 2017006633 W KR2017006633 W KR 2017006633W WO 2017222330 A1 WO2017222330 A1 WO 2017222330A1
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chain
region
amino acid
fviii
coagulation factor
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PCT/KR2017/006633
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French (fr)
Korean (ko)
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강관엽
이승훈
오인재
오미숙
류재환
조의철
이기남
양선아
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재단법인 목암생명과학연구소
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Priority to US16/312,551 priority Critical patent/US20230151078A1/en
Priority to KR1020197002468A priority patent/KR102219859B1/en
Priority to JP2018567079A priority patent/JP7235511B2/en
Priority to EP17815743.4A priority patent/EP3476860A4/en
Priority to CN201780048647.3A priority patent/CN109689683A/en
Publication of WO2017222330A1 publication Critical patent/WO2017222330A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present application is directed to recombinant short chain FVIII protein production techniques and chemical conjugates thereof for use in the treatment of hemophilia.
  • Hemophilia is a disease in which bleeding continues due to lack of coagulation factors.
  • Blood clotting factors consist of I to XIII, of which the factors associated with hemophilia are VIII, IX and XI. Hemophilia is caused by a genetic defect of each of the above factors, and hemoglobin A is classified into hemophilia A lacking Factor VIII, hemophilia B lacking Factor IX and hemophilia C lacking Factor XI, among which hemophilia A is about 80 Account for -85%.
  • hemophilia treatment is hemostasis and treatment is carried out in two ways, either prophylactically or on demand using Enzyme Replacement Therapy (ERT). .
  • ERT Enzyme Replacement Therapy
  • PEGylated FVIII which PEGylates FVIII to reduce binding to low density lipoprotein receptor related protein (LRP1), which is known to be responsible for in vivo clearance of FVIII, thereby preventing loss of FVIII.
  • LRP1 low density lipoprotein receptor related protein
  • FVIII-Fc FVIII-Fc fusion protein fused to the F-terminus of the antibody at the C-terminus of FVIII. Due to circulation in the body by FcRn, the half-life of FVIII is longer than that of FVIII, but FVIII-Fc is Due to the combination with vWF, the residence time in the body was dominated by the half-life of vWF.
  • FVIII-mimetic bi-specific antibody which is an intact IgG form of bispecific antibody containing two types of Fab specific to FIX (or activated FIX) and FX, designed to replace the function of FVIII in the body ( Kitazawa et al, Nature medicine 2012 18 (10): 1570-4.).
  • TFPI anti-issue factor pathway inhibitors
  • Korean Unexamined Patent Publication No. 2014-0114266 discloses anti-hemopathy factor VIII with increased specific activity.
  • the present application seeks to provide short-chain FVIII with improved residence time in the body and easy mass production.
  • the present disclosure includes a heavy region, a light chain and a portion of a B region fragment deleted from human coagulation factor 8 (Factor VIII), wherein the portion of the B region fragment lacking a cleavage site by purine protease.
  • Factor VIII human coagulation factor 8
  • At least 5 amino acids each in the N-terminal and C-terminal directions of the residue, including 1648 and 1649 residues of SEQ ID NO: 1, and partly deleted to include 4 to 6 glycosylation sites Phosphorus, short-chain clotting factor 8 (Factor VIII) is provided.
  • the B region fragment included in the single-chain coagulation factor 8 of the present disclosure includes about 15% to 40% of the wild-type B domain sequence, but is not limited thereto as long as the effect according to the present disclosure is achieved. .
  • Those skilled in the art can select an appropriate single-chain coagulation factor 8 in consideration of the conditions of the B region fragment included in the above-mentioned short-chain coagulation factor 8 described above.
  • the B region is interpreted to include an a3 region as described later.
  • a portion deleted from a region B deleted from a portion that satisfies the above condition may be continuous or discontinuous, and the region B deleted from the region based on the sequence of SEQ ID NO. Based on the sequence of No. 1 (i) amino acid residues 741 to 902 and 1654 to 1689; (ii) amino acid sequences of amino acid residues 741 to 965 and 1654 to 1689; Or (iii) amino acid residues 741-902, 1637-1642 and 1654-1689.
  • the single-chain coagulation factor 8 of the present application includes an amino acid sequence represented by SEQ ID NO: 2 to 7 or a sequence having about 90% or more homology thereto, and the method of determining homology described below. And the disclosure of factors and biological function equality according to the present disclosure may be appropriately determined.
  • the nucleic acid molecule encoding the single-chain coagulation factor 8 having the amino acid sequence represented by SEQ ID NOs: 2 to 7 is represented by SEQ ID NOs: 10 to 15, or the nucleic acid sequence is expressed in a cell. Also included are those substituted with codon optimized sequences or variants with nucleic acid sequences due to degenerate codons.
  • the single-chain coagulation factor 8 according to the present invention is 90% or more of the double-chain coagulation factor inactivation measured by the CS or APTT method.
  • the present invention is conjugated with a hydrophilic polymer, such as a polymer comprising polyethylene glycol, polyethylene oxide, dextran or polysialic, and when PEG is used, it may be via acryloyl, sulfone or maleimide at the conjugation position. Can be spliced.
  • a hydrophilic polymer such as a polymer comprising polyethylene glycol, polyethylene oxide, dextran or polysialic, and when PEG is used, it may be via acryloyl, sulfone or maleimide at the conjugation position. Can be spliced.
  • the conjugated coagulation factor 8 is conjugated with a hydrophilic polymer at some amino acid residues of the A region and / or the B region fragment, and the conjugated position is the sequence of SEQ ID NO: 1 in the B region. At least one position selected from the group consisting of amino acid residues 754, 781, 782, 788, 789, 825 and 897; At least one position selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1 in the A-region, wherein the residue at the conjugated position is for conjugation with the hydrophilic polymer It may be substituted with cysteine.
  • conjugated with PEG in particular PEG with an average molecular weight of at least 20 kDa is used.
  • the application is also directed to the short chain hemagglutination factor 8 according to the invention, in particular to the short chain hemagglutination factor 8 represented by SEQ ID NOS: 2 to 7, or to the region B of the sequence B for conjugation with a hydrophilic polymer in said amino acid sequence.
  • a nucleic acid molecule encoding a single-chain clotting factor 8 containing a cysteine group is provided.
  • the residues into which cysteine is introduced are shown in Table 3, and the nucleic acid molecules whose residues are substituted with cysteine for conjugation with a hydrophilic polymer are represented by SEQ ID NOs: 17 to 32, and corresponding amino acids.
  • the sequence is represented by SEQ ID NOs: 33-48. Also included are those in which the nucleic acid sequence has been replaced with a codon optimized sequence for expression in a cell or the nucleic acid sequence due to degenerate codons is mutated.
  • the present disclosure also provides a vector comprising a nucleic acid molecule according to the present application, a cell comprising the vector.
  • the present invention also provides a method of producing a single-chain blood coagulation factor 8 using a vector or a cell according to the present invention
  • the method according to the present invention comprises the steps of transferring the vector according to the present application to eukaryotic cells, Or optionally providing a cell to which a vector according to the present invention has been delivered: expressing the cells in a single chain FVIII form in which the cysteine introduced through the culture in the culture medium is free thiol group masked by disulfide bonds by cysteine or glutathione. step; Collecting short-chain FVIII expressed from the culture solution and treating it with a reducing agent to release masked cysteine or glutathione; And treating the treated culture solution with PEGylated buffer solution.
  • the present invention also provides a blood coagulation kit for use in a hemophilia patient or any patient in need of blood coagulation comprising any of the short chain coagulation 8 factors disclosed herein, including the claims, hemophilia of the short chain coagulation factor 8
  • a therapeutic coagulation method comprising administering a therapeutic use or composition, or a short-chain coagulation factor 8, to a hemophilia patient or a patient in need of coagulation in a therapeutically effective amount.
  • Single-chain FVIII is a light chain and heavy chain, which is linked to the B-domain lacking some sequences, in particular does not include a cleavage site cleaved by furin protease that occurs during normal FVIII expression process, at least 4 It includes the B region partially deleted to include the site.
  • the short-chain coagulation factor 8 (Factor VIII) or some residues of the A or B region according to the present invention are pegylated short-chain coagulation factor 8, which are prepared in the form of short chains while maintaining the activity thereof, and thus easy to mass-produce.
  • Figure 1 schematically shows a PEGylated short-chain FVIII structure according to an embodiment of the present application, 1648 residues indicate the cleavage position of furin.
  • FIG. 2 schematically shows the production of short chain FVIII from full length FVIII according to one embodiment of the present application.
  • Furin cleavage sites (the 1648 th amino acid sequence based on SEQ ID NO: 1) are indicated and the numbers in the figures represent each region corresponding to the indicated sites as amino acid residues.
  • Figure 3 is a result of Western blot analysis of single-chain FVIII expression according to an embodiment of the present application.
  • FIG 4 shows the PEGylation position tested for optimal PEGylation position of short chain FVIII according to one embodiment of the present disclosure.
  • PK 7 is a test result comparing the pharmacokinetics (PK) of scFVIII with a commercially available FVIII material according to an embodiment of the present application.
  • FIGS. 8A and 8B are schematic diagrams and experimental results for comparing the tail bleeding hemostatic efficacy test of PEGylated scFVIII with the efficacy of commercialized FVIII material, respectively, according to one embodiment of the present application.
  • FIG. 8B PEG40kDa-scFVIII-B3 is shown.
  • the present invention describes the development of a coagulation factor VIII variant that can be expressed in a short chain form with increased productivity and stability in the body, and the coagulation factor of the coagulation factor that improves the convenience of the patient's administration cycle through PEGylation of such a variant. Based on development.
  • Coagulation factor 8 consists of the A1-A2-B-A3-C1-C2 regions and is synthesized as a short-chain protein in hepatocytes. After synthesis, it is processed and matured to form 280kDa heterodimer consisting of heavy and light chains.
  • the light chain is 80kDa and is composed of A3-C1-C2 region
  • the heavy chain is composed of A1-A2-B region
  • the molecular weight is 90-200kDa, which has a large molecular weight difference due to the difference in the length and degree of glycation of the B region.
  • the a1 domain between the A1 and A2 domains and the a2 and a3 domains between the A2 and B domains are included in the heavy chain.
  • This heterodimer is present in the blood in an inactive state associated with vWF, and is cleaved by thrombin behind residues 372, 740 and 1689 upon exposure to stimuli such as vascular damage. As a result, it is separated from vWF and activated to form a trimer of A1, A2 and A3-C1-C2. The trimer then catalyzes the activation of FX by FIXa and is rapidly deactivated.
  • Short-chain FVIII is reported to be structurally stable and to increase expression (see WO2004 / 067566).
  • double-chain type FVIII there was reported a difference in expression according to the length of the B region (Miao et al ., Blood. 2004 May 1; 103 (9): 3412-9.).
  • the present application is a single-chain FVIII in which the heavy and light chain regions of human coagulation factor (Factor VIII) are linked by a B-domain in which some sequences are deleted, in particular the B-domain in which the partial sequences are deleted is a protein. At least 5 amino acids are respectively deleted in the N-terminal and C-terminal directions, respectively, based on the 1648 and 1649 residue positions of SEQ ID NO: 1 so as not to include a cleavage site by a furin, and 4 to And a B region fragment partially deleted to contain six glycosylation sites.
  • Factor VIII human coagulation factor
  • Single chain FVIII comprises an A1-A2-B partially-A3-C1-C2 region.
  • the residue positions and the sequences in FVIII of each region included herein are known and human amino acid sequences such as human FVIII can be represented by the sequence of SEQ ID NO: 1 with heavy chains comprising the A1 and A2 regions. 1-740 residues, the B region is 741-1689 residues, the light chain comprising the A3-, C1, and -C2 region is up to 1690-2332 residues (Fig. 2; and Orlova et al ., Acta Naturae. 2013 Apr- Jun; 5 (2): 19-39.
  • Region B is referred to herein, including the a3 regions 1649-1689 described in FIG. 2.
  • short-chain FVIII may be represented by the formula A1-A2-B'-A3-C1-C2, A1 is the A1 region, A2 is the A2 region, B 'is part of the B region, A3 is A3 region, C1 means C1 region, C2 means C2 region, each of which can be referred to above.
  • the B region (B ′) which is partially deleted in the short chain FVIII according to the present invention is deleted from the protease furin cleavage site and in one embodiment includes 1648 and 1649 residues of SEQ ID NO: 1 and Deletions of at least five amino acids in each of the terminal and C-terminal directions.
  • the deletion region of the B region is determined to include 4 to 6 glycosylation.
  • the B region in which the furin cleavage region included in the short chain FVIII according to the present invention is deleted may be determined in a limit including about 15 to 40% of the total wild type B-domain.
  • deletion site of the B region in which a part included in the short chain FVIII according to the present invention is deleted may be continuous or discontinuous.
  • the FVIII amino acid sequence according to the present disclosure may be represented by SEQ ID NOs: 2 to 7.
  • the recombinant FVIII disclosed herein is not limited to such sequences, but includes biologically equivalents thereof.
  • Biological equivalents are those which have additional modifications to the amino acid sequences disclosed herein, but which have substantially the same activity as the polypeptides according to the invention, such modifications may, for example, delete, insert and / or substitute amino acid sequence residues. It is to include.
  • conservative amino acid substitutions have occurred in a recombinant FVIII polypeptide according to the present disclosure.
  • Conservative amino acid substitutions mean substitutions that do not substantially affect or reduce the activity of a particular polypeptide. For example, 1 to 15 conservative substitutions, 1 to 12, For example, 1, 2, 5, 7, 9, 12, or 15 conservative amino acid substitutions.
  • amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have a similar shape.
  • arginine, lysine and histidine Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.
  • the hydropathic index of amino acids may be considered.
  • Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).
  • hydrophobicity indexes are particularly important for imparting the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions between amino acids which exhibit a hydrophobicity index difference of preferably within ⁇ 2, more preferably within ⁇ 1 and even more preferably within ⁇ 0.5 are advantageous.
  • Amino acid substitutions that do not alter the activity of the recombinant protein according to the present invention are also known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).
  • substitutions are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe And substitutions between Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.
  • sequence comparison methods known in the art. At least 80% homology, in particular at least 85%, more when aligning the sequences disclosed herein to any other sequences as closely as possible and analyzing the aligned sequences using algorithms commonly used in the art. In particular, sequences exhibiting at least 90%, even more particularly at least 95% homology, mean substantial identity. Alignment methods for sequence comparison are known in the art. For example, Smith and Waterman, Adv. Appl. Math. (1981) 2: 482; Needleman and Wunsch, J. Mol. Bio. (1970) 48: 443; Pearson and Lipman, Methods in Mol. Biol.
  • Recombinant single chain FVIII according to the present disclosure is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or as compared to wild-type double-chain FVIII in terms of activity per mole of active substance. It includes those having 100%. Wild-type double-chain FVIII and methods for measuring its activity are well known and can be performed, for example, by determining the inactivation level and (APTT) / CS ratio measured by CS and the clotting assay (APTT: activated partial thromboplastin time). (Coagulation Assays Circulation. 2005; 112: e53-e60).
  • Bi-chain FVIII is processed by purine as described above to consist of dimers of light and heavy chains.
  • recombinant protein of native FVIII Advanced FVIII
  • CHO cells most of which consist of a double-chain containing various lengths of B region, similar to the composition of plasma-derived native FVIII.
  • the double chain FVIII to be compared with the single chain FVIII according to the present application may or may not include the B region.
  • the B region is mainly processed by recombination intentionally removing the B region or expressing a native FVIII and processing the protein and cleavage by FXa (active FX) and thrombin in the blood.
  • FXa active FX
  • thrombin thrombin in the blood.
  • a backbone that does not contain can be generated.
  • the B region may be recombinantly included in part or the whole of the B region, or in the process of expressing a native FVIII, or by the protein cleavage process by FXa (active FX) and thrombin in the blood.
  • FVIII duplexes comprising some or all of may be generated.
  • the double-chain FVIII which is directly compared with the short chain of the present application, expresses recombinant FVIII of a native type including the entire B region by recombinant expression. Subsequently, some or all of the B region is removed by thrombin or FIXa or FXa so that it is a recombinant FVIII that is most similar to the native FVIII in a mixture of FVIII containing no B region or a part of the B region.
  • the present application relates to a nucleic acid molecule or polynucleotide encoding a single-chain recombinant FVIII according to the present application, a vector comprising said polynucleotide or a cell strain transformed with said vector.
  • Nucleic acid molecules encoding the recombinant single-chain FVIII according to the present application includes those that have been codon optimized according to the type of cells expressing the recombinant single-chain FVIII according to the present application.
  • the nucleic acid sequence is an optimization of the nucleic acid sequence encoding the protein of SEQ ID NO: 5 can be represented by the sequence of SEQ ID NO: 15.
  • Nucleic acid molecules encoding recombinant single-chain FVIII according to the present invention also include those encoding FVIII corresponding to substantially the same biological equivalents as described above.
  • nucleic acid molecules encoding a single-chain recombinant FVIII according to the present application can be cloned into various expression vectors for various purposes.
  • the specific configuration of the expression vector may vary depending on the host cell to which the single-chain recombinant FVIII according to the present invention is to be expressed.
  • a vector comprising a nucleic acid molecule encoding a recombinant single chain FVIII according to the present disclosure may be prepared by methods known in the art, for example, a nucleic acid molecule encoding a recombinant single chain FVIII according to the present disclosure may be a promoter and / or Operate connection to the enhancer. In one embodiment is inserted into a recombinant expression vector capable of expressing a foreign gene in the cell, examples of such vectors include, but are not limited to, general protein expression vectors such as pMSGneo, pcDNA3.1 (+).
  • an expression vector including a matrix attachment region (MAR) factor that binds to a nuclear matrix is used.
  • MAR matrix attachment region
  • the MAR factor induces position-independent expression. It plays a role in increasing gene expression. Therefore, when using the pMSGneo vector, a stable and high expression amount can be obtained.
  • pcDNA3.1 (+) vector contains a strong CMV promoter, widely used for protein expression.
  • the recombinant expression vector comprising a nucleic acid molecule encoding a recombinant single-chain FVIII according to the present application can be transformed into a suitable host cell as described below for various purposes.
  • the coding sequence of the nucleic acid molecule encoding the protein can be optimized appropriately for the desired cell.
  • the present application relates to recombinant cells transfected or transformed into a vector according to the present application.
  • Such cells include both prokaryotic and eukaryotic cells that can be used for the amplification of the vector and / or nucleic acid molecules contained in the vector, thereby being used to produce the desired protein.
  • Various cells that can be used for this purpose are known, and those skilled in the art will be able to select appropriate ones in view of the specific purposes and effects herein, and may include, for example, those described in the Examples and the drawings herein, but not limited thereto. It is not. Examples include E. coli, Mammalian cells, Yeast, Plant cells, Insect cells.
  • eukaryotic cells in particular mammalian cell lines, are used for expression of recombinant FVIII.
  • mammalian cell lines include CHO, BHK, COS7, HEK cell lines and the like can be used, and preferably, CHO cell line, in particular CHO-S cell line, CHO-DG44 or CHO-K1 cell line, or HEK cell line especially HEK293 cell line can be used, but not limited thereto.
  • the vector according to the present invention is delivered to such a host cell for expression.
  • Methods for delivering vectors to such host cells are known in the art, for example, calcium phosphate precipitation method, shotgun method, liposome method, using a method known in the art such as nanoneedle or electroporation method May be, but is not limited to such.
  • Short-chain FVIII may be modified at a specific residue with a hydrophilic polymer for the purpose of increasing the half-life.
  • the protein structure of the B region is not known.
  • the B region is a protein with a flexible structure and a high degree of glycosylation, and is known to be rich in negative charge, but its structure is difficult to predict. That is, it is theoretically possible to predict the structure of the B region using molecular modeling techniques, but considering the heterogeneity due to post-translational modification and glycosylation of the B region, it is difficult to predict the B region structure. As such, in order to select a modified position in the B region, structural information on the surface of the B region protein structure is required, but since there is no information about this, it is very difficult to select a PEGylation position.
  • residues of region A and / or B which do not particularly affect the activity of short-chain FVIII are selected through unexpected efforts and modified with hydrophilic polymers to obtain short-chain FVIII with improved half-life while maintaining inherent activity. It was.
  • the PEGylation position was selected between -6 and +6 residues.
  • the present application is conjugated with a hydrophilic polymer at some amino acid residues of the A region and / or B region fragment, and the conjugated position is based on the amino acid residues 754, 781, 782 in the B region based on the sequence of SEQ ID NO: 0. At least one position selected from the group consisting of 788, 789, 825 and 897; At least one position selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 0 in the A-region, wherein the residue at the conjugated position is for conjugation with the hydrophilic polymer A single-chain coagulation factor 8 is substituted with cysteine.
  • Hydrophilic polymers that can be used for the modification of short-chain FVIII according to the present application can be used a variety of polymers known in the art, including, but not limited to, polyethylene glycol, polyethylene oxide, dextran, polysialic, etc. It is not. These polymers are biocompatible (non-toxic, low-toxic) hydrophilic, flexible structures that prevent the loss of activity and prevent loss of activity due to non-specific interactions of the conjugated proteins with chemically conjugated proteins. It serves to prevent inactivation by proteolytic enzymes.
  • PEG is used, in particular PEG of at least 20 kDa.
  • PEG binds to proteins and gives them the effect of wrapping around the protein, which can then be lost after binding to scavenger receptors in the body or to prevent degradation by inactivating protease.
  • PEG is a chain-shaped polymer that is linear or branched, and small molecular weight PEG does not cover the protein enough to completely protect the protein. Therefore, the molecular weight must be above the limit level to sufficiently cover the protein to be protected.
  • PEGylation of the short-chain FVIII of the present application may be conjugated via PEG containing one end of an acryloyl, sulfone or maleimide group specific to cysteine after substitution of an amino acid at the PEGylation position with cysteine.
  • the short-chain FVIII according to the present application may be substituted with a specific residue for modification to the hydrophilic polymer, specifically, the residue to be replaced depends on the position where the hydrophilic polymer is conjugated on the short-chain FVIII, and those skilled in the art May be modified with appropriate residues.
  • PEGylation is used as the modification, in which case the residue to be modified is substituted with cysteine.
  • the residue is substituted with cysteine for PEGylation.
  • a protein substituted with cysteine for modification can be performed using methods known in the art, for example, for proteins to be PEGylated, free cysteine introduced is free during expression in cells or after exiting cells.
  • free cysteine introduced by forming disulfide bonds with cysteine or glutathione, a low-molecular substance containing thiol, and stabilizing the free cystine introduced by reducing conditions immediately before PEGylation to increase PEGylation efficiency need.
  • the PEGylation reaction can be carried out using a known method, for example, cysteine or glutathione used for masking free cysteine must be removed before the PEGylation reaction to restore the free thiol of cystine introduced. , Including the treatment of reducing agents such as DTT, beta-mercaptoethanol, cysteine, glutathione (reduced form), and oxidation to restore the reductive cleavage of the native disulfide bonds in this process. Can be.
  • cysteine or glutathione used for masking free cysteine in addition to the cell culture medium components or
  • cysteine or glutathione components which are normally produced, accumulated and secreted by cells during growth and maintenance of cells, may be used without additional addition to cell culture medium components.
  • the present invention also provides a method of transfecting a vector according to the present application into an eukaryotic cell; Culturing the cells in culture; Collecting the culture solution to purify cysteine-introduced FVIII; And treating the purified cysteine introduced FVIII with PEGylation buffer; Restoring the introduced free thiol group of cysteine through a reduction process; Oxidizing to restore disulfide bonds within the isolated short chain FVIII protein during the reduction process;
  • the present invention relates to a method for producing short-chain coagulation factor 8, comprising specifically PEGylating a short-chain FVIII cysteine restored to a reduced thiol group and isolating PEGylated short-chain FVIII.
  • Recombinant single-chain FVIII according to the present invention can be usefully used for the treatment of hemophilia A, hemophilia, in particular hemophilia A patients, or hemophilia A.
  • the present application also provides a blood coagulation kit for use in a hemophilia patient or a patient in need of blood coagulation, including any one of the short chain coagulation factors 8 disclosed herein, including the claims of the present application, Use or composition for the treatment of hemophilia, or a composition comprising a nucleic acid of a base sequence encoding a single-chain coagulation factor 8 Nucleic acid of the hemophilia type A using the composition and the nucleic acid of the base sequence encoding a single-chain coagulation factor 8 to be used in the treatment It relates to a hemophilia treatment method or a blood coagulation method comprising the step of administering to the hemophilia patients or patients in need of coagulation of the blood containing a composition or a short-chain coagulation factor eight.
  • Recombinant single-chain FVIII according to the present application may be provided in the form of a pharmaceutical composition for treating hemophilia comprising a pharmaceutically acceptable carrier.
  • composition of the present invention can be used alone or in combination with methods using other drug treatments and biological response modifiers.
  • composition of the present invention may be prepared by including one or more pharmaceutically or physiologically acceptable carriers in addition to the above-mentioned active ingredients.
  • carrier means a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to a cell or mammal that is exposed to the dosages and concentrations employed.
  • examples of such carriers include saline, Ringer's solution, buffered saline, buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin Or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, for example EDTA, sugars Alcohols such as mannitol or sorbitol, salt-forming counter ions such as sodium, and / or nonionic surfactants such as tween, polyethylene glycol (P
  • injectable formulations such as aqueous solutions, suspensions, emulsions and the like.
  • injectable formulations such as aqueous solutions, suspensions, emulsions and the like.
  • it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.
  • compositions herein are particularly preferred parenteral administration (eg, intravenous, subcutaneous, intraperitoneal) according to the desired method.
  • the dosage depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the time of day, and may be appropriately selected by those skilled in the art.
  • composition according to the invention is administered in a therapeutically effective amount.
  • therapeutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with an effective dose level being the type of disease, severity, activity of the drug, drug Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.
  • the compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • Recombinant single-chain FVIII according to the present application may be administered in a therapeutically effective amount to a subject in need of hemophilia or coagulation in the form of a composition as described above or in the form of a blood coagulation method or a method of treating hemophilia, This may be referred to the above description.
  • treatment means any action in which symptoms caused by a disease are improved or beneficially altered by administration of a composition of the present invention.
  • Subjects in which the methods according to the invention are used are primates including but not limited to humans.
  • FVIII expressed in the short chain form
  • FVIII was constructed in which a part of the B region including the furin cleavage position 1648 residue (based on the amino acid sequence of SEQ ID NO: 0 representing the full length FVIII amino acid sequence) was deleted. Since the length and length of the B region connecting the heavy and light chains may affect the activity and properties of the final FVIII, a total of seven types of FVIII were constructed as shown in Table 1 to determine the length of the B region included in the scFVIII. Each scFVIII constructed was constructed to include a B region of a certain length from the N-terminal (SEQ ID NO: 741 amino acid residue) of the B region and to contain the sugar chain originally possessed by this B region.
  • a portion of the a3 domain adjacent to the light chain site is contained more than scFVIII of other structures.
  • SEQ ID NO: 6 scFVIII optimized with CHO codon was made to enhance expression in CHO cells (nucleic acid SEQ ID NO: 15).
  • Each scFVIII lacking the B region was synthesized based on the human FVIII nucleic acid sequence (NM000132).
  • the G1 protein sequence (Leader-Heavy chain-B domin (741-764, 1653-1689) -Light chain) was encoded based on the human FVIII nucleic acid sequence (NM000132) to prepare the scFVIII gene expression vector.
  • Genes were synthesized through GeneArt to include Xho I- Pac I enzyme cleavage sites in Not I- Asis I and 3 '.
  • the synthesized gene was cloned into the Not I / Xho I site of the pcDSW vector to construct a pcDSW-G1 expression vector, and the accuracy of the expression vector construction was confirmed by comparing the cut band sizes after cleavage using the Asis I / Pac I enzyme. It was.
  • Each scFVIII (G2, G3, G4, G4-flex, G6) is deleted from the BamH I site (GGATCC) in the A2 region of the FVIII heavy chain from the gene chain to include the Pac I enzyme cleavage site at the end of the light chain.
  • Example 1-1 ScFVIII constructed in Example 1-1 was expressed in cells, and the expressed cells were cultured in cell culture medium.
  • the expression vector constructed in Example 1-1 was transfected into Expi293F TM cells using the transient expression system Expi293F TM Expression System Kit (Thermofisher, Catalog Number A14635). Briefly, Expi293F TM cells were passaged at 2.0 X 10 6 cells / mL at an expected requirement 24 hours before transformation, and then the number of cells and viability were measured on the day of transformation. When cell viability was 95% or more, transformation was performed.
  • 7.5 X 10 7 cells were added to a 125 mL flask to adjust the final volume of 25.5 mL by adding Expi293 culture medium (based on 30 mL).
  • 30 ⁇ g of the expression vector constructed in Example 1-1 was mixed using Opti-MEM so that the total volume was 1500 ⁇ L.
  • 80 ⁇ L transfection reagent was mixed to a total volume of 1500 ⁇ L using Opti-MEM and incubated at room temperature for 5 minutes. After 5 minutes, the Opti-MEM containing the transfection reagent was added to the Opti-MEM containing the DNA and mixed gently. And reacted at room temperature for 20-30 minutes.
  • a 3 mL DNA: transfection reagent complex was previously dropped in a 125 mL flask Expi293F TM cell (Total volume: 28.5 mL) and incubated at 37 rpm, 125 rpm in a 5% CO 2 shacking incubator. After 16 to 20 hours, 150 ⁇ L and 1.5 mL of Enhancer 1 and Enhancer 2 were added thereto, and then incubated at 34 rpm and 125 rpm in a 5% CO 2 shacking incubator. On day 3 of culture, the medium was harvested and scFVIII activity was measured as follows.
  • BDD-FVIII was up to 50% lower in activity as measured by coagulation assay (APTT) than in chromogenic assay (CS), while full-length FVIII was similar in both measurements (BARROWCLIFFE et al., SEMIN THROMB HEMOST, vol. 28 (3), 2002: 47-56).
  • the low activity of BDD in clotting assays has been attributed to the fact that BDD-FVIII does not prevent bleeding in patients with hemophilia A than full-length FVIII in real-term prophylaxis (GRUPPO, RA, et al., 2003.
  • the FVIII activity measurement test methods used herein are two-stage method and chromogenic method.
  • One-stage method is a method for measuring FVIII activity of a sample by mixing FVIII deficient plasma, activator, phospholipids and FVIII sample and measuring blood coagulation time.
  • the chromogenic method mixes FIXa, FX, thrombin, calcium, phospholipids and FVIII samples, inserts a chromogenic substrate that is cut and developed by FXa to measure the amount of FXa activated by the FVIII sample, and develops the chromogenic substrate. It is a method of measuring the activity of FVIII according to the degree.
  • the one-stage method uses FVIII deficient plasma and measures clotting time.
  • the chromogenic method does not use FVIII deficient plasma, but the factors necessary for activation of FVIII and activated FVIII. After adding the substrate for measurement, the degree of color development of the substrate is measured.
  • Activity measurement by the one-stage method was performed using an automated method previously prepared on the ACL TOP CTS 500 instrument as follows. Samples were diluted to 1 IU / mL concentration using FVIII deficient plasma, and 1 mL distilled water was dissolved in NIBSC FVIII standard vial, and then diluted to 1 IU / mL concentration using FVIII deficient plasma. Consumable reagents and measurement samples were prepared according to the rack type of the experimental equipment. The calcium chloride vial was placed in the R rack of the ACL TOP CTS 500 equipment in order to recognize the barcode, and the magnetic stirring bar was placed in the APTT-SP vial and placed in the R rack so that the barcode of the vial was recognized.
  • FVIII deficient plasma was prepared by measuring the number of samples ⁇ 400 ⁇ L and taped the periphery to Ral to prevent vial recognition.
  • DA rack has G.O.
  • a vial containing 2.63 mL of albumin was placed in 50 mL of buffer (Imidazole 3.4 g, NaCl 5.8 g, 1 L, pH 7.4). Put the sample prepared above and 400 ⁇ L into the sample cup and place the sample cup in the sample rack.
  • Information from unrecognized reagents and measurement samples was entered in the ACL TOP program, and pre-made automated assays were run. All reactions of the automated assay proceeded as follows at 37 degrees.
  • the sample to be measured is pre-dilution 10 times with G.O buffer, and then diluted 1, 3, and 10 times further to make 100%, 33,33%, and 10% samples.
  • 50 ⁇ L of the diluted sample was allowed to stand for 30-45 seconds, and then mixed with 50 ⁇ L FVIII deficient plasma and allowed to stand for 60-70 seconds.
  • 50 ⁇ L (intermediate reagent) APTT-SP the mixture was allowed to stand for 300-340 seconds, and finally, 50 ⁇ L start reagent (APTT-SP CaCl 2) was added, and the solidification time was measured.
  • Chromogenic method was used to measure the activity of chromogenic assay kit sold by CHROMOGENIX as an endpoint method, and the test method provided by the manufacturer was modified to suit the present test as follows. Specifically, dilute the sample to enter the standard range (0.25-1 IU / mL) using a 1 ⁇ dilution buffer. Calibration plasma was prepared using a 1 ⁇ dilution buffer to a concentration of 0, 0.25, 0.5, 1 IU / mL. 10 ⁇ L of the prepared sample and 10 ⁇ L of the standard product were diluted with 790 ⁇ L of 1 ⁇ dilution buffer, and then 50 ⁇ L was dispensed into a 96 well plate and left at 37 ° C. for 5 minutes.
  • 50 ⁇ L of the reagent reagent was dispensed into each well by using an automatic dispenser pipette, followed by reaction at 37 ° C. for 2 minutes.
  • 50 ⁇ L substrates were dispensed into each well and allowed to develop for 2 minutes at 37 ° C. for 2 minutes.
  • 50mL 2% citric acid was added to the developed sample to stop the reaction. After measuring the absorbance at 405nm wavelength to draw a linear calibration curve, the absorbance of the sample was substituted into the calibration curve to measure the activity of the sample.
  • scFVIII expressed in the culture medium increased with the increase in the length of the B region, and it was confirmed that the expression level was significantly increased in scF4, scF5, scF6, scF7 having a length of the B region of 198 or more amino acids.
  • scF3, scF4, scF5, scF6, and scF7 which have a length of 92 or more amino acids, showed similar levels of inactivation to the recombinant full-length FVIII (Advate®, manufactured by Baxalta) and used as a reference. Similar to Advate® used as reference.
  • the expression level was significantly lower, and the length of the B region was shorter than the length of 84 amino acid protein sequences such as scF1, scF2 of the Example. Inactivity was significantly lower than Advate®, the full-length FVIII used as a reference.
  • Short-chain FVIII is activated by thrombin cleavage at the site comprising the B region connecting the heavy and light chains in the activation step. If the length of the B region, which is the linking site between the heavy and light chains, is short, the structural limitation by the short linker length may affect the activity due to the abnormal conversion of short-chain FVIII to thrombin-activated FVIII. However, in the case of the double-chain FVIII, there is no structural limitation, and thus activation of thrombin regardless of the length of the B region is expected to be smooth. It can be seen from the similarity to the activity of FVIII.
  • the length and sequence of the B region of the single-chain FVIII are selected so as not to affect thrombin activation, so that the thrombin activation sites (1689-1690 and 740-741; ) was activated similar to the native (ie wild-type) FVIII, yielding similar short-chain FVIII in addition to the full-length FVIII used in comparison.
  • the culture solution and LDS sample buffer were mixed at a ratio of 3: 1, respectively, and loaded with 30 ⁇ L of the sample mixed in 4-12% bis-tris (BT) gel, and then run at 150 volt for about 1 hour. After running the gel was transferred to Nitrocellulose (NC) membrane, it was allowed to stand for one hour in blocking buffer (Thermo Scientific). Subsequently, primary antibodies that recognize heavy chains (GMA-012, Green Mountain Antibodies) and light chains (ab41188, Abcam) of FVIII were prepared by diluting them in TBST buffer, and then placed in a blocked NC membrane and reacted for 1 hour. Was performed.
  • the stability of scF1, scF2, and scF3 having a relatively short B region was superior to scF4, scF5, scF6, and scF7, but the expression level was relatively low or inactive.
  • short-chain FVIII having various lengths of B region derived from natural type double-chain FVIII was prepared and characterized, and the effect of length of B region on the expression level, inactivation, and stability was evaluated to evaluate the effect of FVIII and activity of natural type. Recombinant single-chain FVIII with similar expression characteristics and high expression level could be produced.
  • the sequence and length of the B region in the single-chain FVIII including a part of the B region may affect the FVIII activity characteristics, the activity and properties of the recombinant blood coagulation FVIII may be affected.
  • the PEGylation site was selected to give persistence in the body based on the scFVIII produced in Example 1, which is composed of the B region of the sequence and the length to preserve the properties.
  • PEGylation was performed at various positions in the B region. Because PEGylation targets the surface residues of proteins, the three-dimensional structure of the B region is required to select the PEGylation position. However, since the structure is unknown, the PEGylation position was difficult to select.
  • the PEGylation position of the B region selected herein selectively PEGylated around the sugar chain of the B region. In other words, the sugar chain is located outside the protein structure, and residues around the sugar chain of the B region are always likely to go out, and thus it may be advantageous for pegylation.
  • cysteine is located at various positions near the sugar chain of the B region based on G4 constructed in Example 1 as shown in Table 3 and FIG. 4, that is, between +6 and -6 residues based on the N-sugar chain.
  • the introduced recombinant scFVIII variant was constructed and subjected to PEGylation selectively to the introduced cysteine. This PEGylation has been described for G4, single-chain FVIII, but can also be applied to the PEGylation of other scFVIII containing sugar chains constructed herein with the same logic.
  • Example 1-1 An expression vector prepared in Example 1-1 after synthesizing a gene through GeneArt to include a cysteine substitution site and a BamH I site (GGATCC) present in the A2 region of the FVIII heavy chain, and a light chain and a Pac I enzyme cleavage site.
  • pcDSW-scFVIII G4 was removed using the enzyme BamH I / Pac I, and the cloned gene was cloned into BamH I / Pac I site to clone the synthesized pcDSW-scFVIII G4 (B1, B2, B3, B4, B5, B6, B7, A3-1 or 4L) expression vectors were constructed and the accuracy of expression vector construction was confirmed by sequencing.
  • cysteine is introduced scFVIII G4 (A2-1, A2-2, or A2-3) are present in the Kpn I Asis I enzyme cleavage site from the area A2 present in the A1 region of the FVIII heavy chain comprising a cysteine substitution site site (GGTACC) by using the GeneArt Inc.
  • Transient expression was performed in the same manner as in Example 1-2 using the mscFVIII expression vector into which the constructed cysteine was introduced.
  • the amount of expression of each cystine introduced FVIII is shown in Table 3 below.
  • Table 3 the scFVIII variant in which cysteine was introduced around the glycan chain in the B region was normally expressed in the culture medium.
  • the expression level was somewhat lower than scFVIII prior to cysteine introduction, which is thought to be because cysteine introduced into scFVIII affects the expression process and stability of scFVIII in culture.
  • PEGylation of the scFVIII variant into which Cysteine was introduced as in Example 2-1 to screen for PEGylation sites was performed as follows. Concentrate 50 mL of the cell culture with 5 kDa MWCO (molecular weight cutoff) and add 5 M NaCl conc solution to the final 1 M NaCl.
  • V8 select resin GE
  • equilibration buffer 20 mM Histidine, 500 mM NaCl, 0.1% (w / v) Tween®80, pH 7.0
  • the concentrated sample were thoroughly stirred together and washed with equilibration buffer
  • elution buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w / v) Tween®80, pH 7.0, 50% (v / v) propylene glycol).
  • the eluted samples were exchanged with pegylation buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w / v) Tween®80, pH 7.0, 5% (w / v) sucrose) using a 5kDa MWCO membrane, followed by a final 0.2 mM of TCEP. Treated and reacted at 4 degrees for 1 hour.
  • Cysteine-introduced scFVIII variants require cysteine and glutathione to be masked for pegylation because cysteine introduced during cell culture is masked as a disulfide bond by cysteine or glutathione.
  • TCEP tris (2-carboxyethyl) phosphine
  • PD10 desalination column
  • maleimide-PEG specific for cysteine (40 kDa) was treated to pegylated.
  • SDS PAGE was performed on the PEGylation reaction to analyze the degree of PEGylation.
  • pegylated scFVIII was produced at the position selected in Example 2 as follows.
  • the cysteine constructed in Example 2-1 introduced scFVIII G4 (B3) and scFVIII G4 (A2_1) were expressed in each cell and cultured.
  • the expression vector constructed in Example 2-1 was transferred to Expi293F TM cells using an Expi293F TM Expression System Kit (Thermofisher, Catalog Number A14635) according to the manufacturer's method.
  • the cell number and viability at the time of transformation were measured and transformation was performed when the cell viability was 95% or more.
  • Into a 125mL flask was adjusted to 25.5mL by adding Expi293 culture medium to 7.5 X 10 7 cells.
  • 30 ⁇ g of the expression vector constructed in Example 2-2 was mixed using Opti-MEM so that the total volume was 1500 ⁇ L.
  • 80 ⁇ L transfection reagent was mixed to a total volume of 1500 ⁇ L using Opti-MEM and incubated for 5 minutes at room temperature. After 5 minutes, the Opti-MEM containing the transfection reagent was added to the Opti-MEM containing the DNA and mixed gently. And reacted at room temperature for 20-30 minutes.
  • a 3 mL DNA: transfection reagent complex was previously dropped into a 125 mL flask Expi293F TM cell (Total volume: 28.5 mL) and incubated at 125 rpm in a 37 ° C., 5% CO 2 shacking incubator. After 16-20 hours, 150 ⁇ L and 1.5 mL of Enhancer 1 and Enhancer 2 were added thereto, and the cells were incubated at 125 rpm in a 34 ° C., 5% CO 2 stirred incubator. On the second day of culture, all the cells were centrifuged to completely remove the existing culture medium.
  • the PEGylation and purification process is largely composed of five stages, and purification was performed by pooling the culture medium 3, 4, and 5 in Example 3-1.
  • the VIIISelect resin developed by GE for the purification of FVIII was used to isolate and purify FVIII from the culture.
  • the equilibration buffer (20 mM Histidine, 5 mM CaCl 2 , 1 M NaCl, 0.02% Tween® 80, pH 7.0) was run out to equilibrate. 5M NaCl buffer was added to the final culture solution to the final 1M NaCl concentration, the pH was adjusted to 7.0, and then loaded on the column. After incubation, the equilibration buffer was equilibrated by flowing until UV dropped to the baseline.
  • Elution buffer (20 mM Histidine, 5 mM CaCl 2 , 0.9 M Arginine, 45% propyleneglycol, 0.02% Tween®80, pH 6.5) was run out to elute scFVIII.
  • GE's SP fast flow resin was used to rapidly remove propylene glycol in the eluate of the first step.
  • wash the column by flowing washing buffer 0.5M NaOH, 1M NaCl.
  • Equilibration was performed by flushing equilibration buffer (20 mM Histidine, 5 mM CaCl 2 , 0.02% Tween® 80, pH 7.0).
  • the eluate of the VIIISelect process was diluted 10-fold with equilibration buffer, then the pH was titrated to 7.0 and loaded onto the column. After loading, the equilibration buffer was equilibrated by flowing until UV dropped to the baseline.
  • Elution buffer (20 mM Histidine, 5 mM CaCl 2 , 400 mM NaCl, 0.02% Tween® 80, pH 6.5) was run out to rapidly elute scFVIII.
  • TCEP was added to the purified scFVIII solution so that the final concentration was 0.1 mM, and the cysteine was reduced by standing at 4 ° C for 1 hour. Residual TCEP was removed using a PD-10 column (GE healthcare) equilibrated with PEGylation buffer (20 mM Histidine, 5 mM CaCl 2 , 200 mM NaCl, 0.02% Tween® 80, pH 7.0). In order to oxidize the disulfide bond of the reduced FVIII itself, it was left at 4 ° C for 2 hours.
  • PEG conjugation of scFVIII G4 was allowed to stand for 12-16 hours at 4 ° C after adding 40 kDa branched methoxy maleimide PEG solution (50mg / mL) dissolved in DMSO so that the PEG ratio per protein was 1:20. .
  • the scFVIII G4 (A2_1) PEG conjugation was performed in the same manner using a 60 kDa branched methoxy maleimide PEG.
  • GE's SP fast flow resin was used to remove scFVIII without PEG and scFVIII with two or more PEGs, impurity generated during PEGylation.
  • the column was washed by flowing washing buffer (0.5M NaOH, 1M NaCl). Equilibration was performed by flushing equilibration buffer (20 mM Histidine, 5 mM CaCl 2 , 0.02% Tween® 80, pH 7.0). The eluate of the PEGylation process was diluted 10-fold with equilibration buffer, then the pH was titrated to 7.0 and loaded onto the column.
  • the equilibration buffer was equilibrated by running until UV fell to the baseline.
  • NaCl concentration in elution buffer (20 mM Histidine, 5 mM CaCl 2 , 50-400 mM NaCl, 0.02% Tween®80, pH 6.5) was stepped up, and PEG-scFVIII was eluted selectively.
  • PEG-scFVIII eluted in the fourth step was concentrated.
  • the concentration was 25 IU / mL using Millipore's amicon 30kDa.
  • the activity of the purified PEGylated scFVIII (PEG-scFVIII-B3, PEG-scFVIII-A2-2) used in Example 3-2 was measured by CS and APTT methods.
  • the CS test method was performed in the same manner as described in Example 1-2, and the APTT test method was performed by modifying the method described in Example 1-2.
  • the activity of PEGylated FVIII was measured by the APTT method, the colloidal silica-based APTT-SP was used as an activator, and the lower activity than the original activity was analyzed.
  • the ellagic acid synthAFax (IL) was used, the normal activity was measured. (Gu. JM et al., 2014.
  • PEG-scFVIII-B3 and PEG-scFVIII-A2-2 showed similar APTT / CS ratio (APTT / CS) to Advate®, which is a two-chain FVIII containing full-length B region as a comparative material. It was confirmed that Xyntha, which is a double-chain FVIII from which the B region was removed, and the APTT / CS ratio were different. This indicates that the scFVIII protein produced herein did not result in a change in the activity properties of blood coagulation FVIII by short chain and PEGylation, considering that the activity of the native FVIII was almost the same in CS and APTT.
  • Xyntha is a double-chain recombinant FVIII in which the B-domain is completely removed.
  • coagulation activity APTT
  • PEG-scFVIII-B3 and PEG-scFVIII-A2-2 maintain blood coagulation activity even after short-chain and pegylation, these two substances according to the present application are considered to have advantages as therapeutic agents for hemophilia.
  • the half-life of pegylated scFVIII was distributed at the University of Pennsylvania, and it was compared and compared in 11-12-week-old hemophilia type A mouse model (B6; 129S4-F8tm1Kaz / J (FVIII-knock-out)) identified by genotyping. Compared with Advate®.
  • the test substance was G4, scFVIII, PEG40kDa-G4scFVIII-B3 (see Example 3), which was site-specific PEGylated with 40kDa branched PEG maleimide after substitution of cysteine with 782 isoleucine at the B region of G4 (see Example 3), 495 of the A2 region of G4.
  • the first valine is cy60e-substituted PEG60kDa-G4scFVIII-A2-2 with 60kDa branched PEG maleimide and Advate®, a recombinant FVIII of the double chain.
  • test animals used for pharmacokinetics were hemophilia mice (females) administered to the tail vein at 125 IU / kg in three hemophilia mice per sample.
  • Blood was collected from the orbit by time (5min, 4hr, 8hr, 16hr, 24hr, 32hr, 40hr, 48hr, 56hr, 72hr) after administration and treated with 10% anticoagulant (3.2% sodium citrate), followed by centrifugation. Separated.
  • the activity of FVIII present in plasma was measured using CS assay measurement reagents as described in Examples 1-2.
  • the profile of the average of FVIII activity in plasma collected from mice of each sample group by time is shown in FIG. 7.
  • the PK parameter analysis was performed using the NCA (Non Compartmental Analysis) method using the WinNonlin software to calculate the mean and standard deviation of plasma FVIII CS activity.
  • NCA Non Compartmental Analysis
  • WinNonlin software to calculate the mean and standard deviation of plasma FVIII CS activity.
  • the half-life of drugs was found in the order B3 ⁇ A2-2> G4> Advate® (Table 5). Judging from the mean data, the half-life of drugs increased by 1.9 times for B3, 1.8 times for A2-2, and 1.2 times for G4 (Table 5).
  • mice The coagulation effect of tail bleeding in mice was measured as follows.
  • Mouse was anesthetized by intraperitoneal administration of Entobarbital Sodium at a concentration of 60 mg / kg.
  • the test substance dose was calculated relative to the mouse body weight (mouse weight was between 19-25 g), and then the test substance was administered intravenously with jugular vein. After 5 minutes of administration, the mouse tail was cut 4 mm from the tip, and immediately after 2 cm of the tail was immersed in a conical tube containing saline, blood was collected for 30 minutes.
  • Test substances were compared to Advate® with PEG-scFVIII-B3 prepared in Example 3. Dose (dosage and dose volume) and distribution of mice in each test substance group are shown in Table 6 below.
  • the blood collected for each mouse was centrifuged at 1500 x g for 5 minutes to remove the supernatant, and then the 10 mL pipet was used to add 3 mL of distilled water to the conical tube to 10 mL, and the blood was completely hemolyzed using vortex. Blood loss measurement was performed according to the manual provided in the Hemoglobin assay kit (sigma, MAK115-1KT).
  • the blood loss values of 40kDaPEG-G4scFVIII-B3 and Adate 50, 100, 200 IU / kg group were significantly different from those of HA group, and 40kDaPEG-G4scFVIII- in HA mouse.
  • the acute tail-clipping test showed that both test substances reduced the amount of blood loss in a concentration-dependent manner (see FIG. 8B).
  • scFVIII in order to select scFVIII having excellent expression, retaining and maximally retaining natural FVIII activity and preventing degradation in culture, scFVIII including B regions of various lengths and sequences were prepared and expressed, and then the activity was measured. Through the process of confirming the stability, scFVIII was developed which has useful properties as a hemophilia type A therapeutic agent. In addition, various PEGylated conjugates were prepared based on the scFVIII developed in the present application, thereby improving FVIII PEGylated conjugates having improved usefulness as therapeutic agents.

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Abstract

The present application provides a single-chain blood coagulation factor VIII including a B region which is partially deleted to contain at least four glycosylation sites, but not a heavy chain, a light-chain, and a region to be cleaved by proteases, or a single-chain blood coagulation factor VIII in which the A or B region has some residues pegylated. A single-chain blood coagulation factor VIII according to the present application not only retains intact therapeutic efficacy and can be easily produced on mass scale because of its single chain form, but also has an increased half-life in vivo through pegylation, thus enhancing convenience for patients as a therapeutic agent for hemophilia A and bringing about a reduction of medical expenses through reduction in production cost.

Description

재조합 단쇄 FVIII 및 그 화학 접합물Recombinant Single Chain FVIII and Chemical Conjugates thereof
본원은 혈우병의 치료에 사용되는 재조합 단쇄 FVIII 단백질 제조 기술과 그 화학 접합물에 관한 것이다. The present application is directed to recombinant short chain FVIII protein production techniques and chemical conjugates thereof for use in the treatment of hemophilia.
혈우병은 혈액 응고인자의 부족으로 지혈이 되지 않아 출혈이 지속적으로 생기는 질환이다. 혈액 내 응고인자는 I~XIII으로 구성되어 있으며, 이중 혈우병과 관련 있는 인자는 VIII, IX 및 XI이다. 혈우병은 상기 각 인자의 유전자 결함으로 발생되며, 부족한 응고인자의 종류에 따라 Factor VIII이 부족한 혈우병 A, Factor IX이 부족한 혈우병 B 및 Factor XI이 부족한 혈우병 C로 구분되며, 그 중 혈우병 A가 약 80-85%를 차지한다. Hemophilia is a disease in which bleeding continues due to lack of coagulation factors. Blood clotting factors consist of I to XIII, of which the factors associated with hemophilia are VIII, IX and XI. Hemophilia is caused by a genetic defect of each of the above factors, and hemoglobin A is classified into hemophilia A lacking Factor VIII, hemophilia B lacking Factor IX and hemophilia C lacking Factor XI, among which hemophilia A is about 80 Account for -85%.
혈우병 치료의 목적은 지혈이며 치료는 효소대체요법(ERT, Enzyme Replacement Therapy)을 이용하여 예방적 또는 대증적(on demand)의 두 가지 방법에서 수행되는데, 현재 치료법은 예방적 측면으로 옮겨가는 추세이다. The purpose of hemophilia treatment is hemostasis and treatment is carried out in two ways, either prophylactically or on demand using Enzyme Replacement Therapy (ERT). .
ERT를 위해 사용되는 단백질 치료제로 과거에는 인간 유래 전혈과 혈장에서 추출하여 농축한 FVIII(혈우병 A), FIX 인자(혈우병 B)가 사용되었다. 하지만 인간 유래 원료 사용 인한 부작용과 합병증의 문제가 발생하였다. 따라서 최근에는 유전자 재조합 기술로 생산된 다양한 인자가 개발되어 사용되고 있다. FVIII 역시 유전자 재조합 기술로 생산되어 감염증과 합병증의 위험을 많이 낮추었으나 짧은 반감기로 인하여 환자에게서 자주 투여해야만하는 불편함을 안고있다. 그래서 다양한 체내 지속형 FVIII을 개발하여 환자의 삶의 질을 향상시키고자하는 노력이 계속되어왔다. In the past, protein therapeutics used for ERT have been used in humans derived from whole blood and plasma and concentrated in FVIII (hemophilia A) and FIX factor (hemophilia B). However, there were problems of side effects and complications due to the use of human-derived ingredients. Therefore, recently, various factors produced by genetic recombination technology have been developed and used. FVIII is also produced by genetic recombination technology, which greatly reduces the risk of infection and complications, but suffers from the inconvenience of having to be frequently administered to patients due to its short half-life. Therefore, efforts have been made to improve the quality of life of patients by developing various in-vivo type FVIII.
그 중 하나는 페길화된 FVIII로 FVIII의 생체내 제거(clearance)를 담당하는 것으로 알려진 LRP1(Low density lipoprotein receptor related protein)과의 결합을 낮추기 위하여 FVIII을 페길화한 것으로 이에 의해 FVIII의 소실이 방지되어 체내 반감기가 어느 정도 향상되었으나 기대에 못 미치는 수준이다. FVIII은 vWF와의 상호작용이 강하여 체내에서 대부분의 FVIII은 vWF와 결합한 상태로 존재하고 따라서 FVIII의 반감기가 FVIII에 결합되어 있는 vWF의 체내 반감기에 의해 결정된다. 그리고 이러한 상황은 페길화 FVIII에도 그대로 적용되어 페길화 이후 페길화 FVIII의 반감기 역시 vWF의 반감기에 의해 지배되어 반감기 향상이 기대 수준에 못 미치는 제한이 있었다.One of them is PEGylated FVIII, which PEGylates FVIII to reduce binding to low density lipoprotein receptor related protein (LRP1), which is known to be responsible for in vivo clearance of FVIII, thereby preventing loss of FVIII. As a result, the half-life of the body improved to some extent, but it was not as expected. FVIII has a strong interaction with vWF, so most of the FVIII in the body remains bound to vWF, and therefore the half-life of FVIII is determined by the half-life of vWF with FVIII bound. This situation also applies to pegylated FVIII, and the half-life of pegylated FVIII after pegylation is also dominated by the half-life of vWF, which limits the improvement in half-life.
다른 하나는 FVIII의 C-말단에 항체의 Fc 영역을 융합한 FVIII-Fc 융합 단백질(FVIII-Fc)로, FcRn에 의해 체내에서의 순환으로 인하여, FVIII 보다 체내 반감기가 길지만 FVIII-Fc 역시 체내에서 vWF와 결합하므로 체내 체류시간이 vWF의 반감기에 의해 지배 받는 문제점이 있었다. The other is the FVIII-Fc fusion protein (FVIII-Fc) fused to the F-terminus of the antibody at the C-terminus of FVIII. Due to circulation in the body by FcRn, the half-life of FVIII is longer than that of FVIII, but FVIII-Fc is Due to the combination with vWF, the residence time in the body was dominated by the half-life of vWF.
이외에 혈우병 A 치료를 위해 기존의 FVIII이 아닌 다른 경로를 통해 FVIII의 혈액 응고인자로서의 기능을 대신하고 체내 지속 효과와 환자의 편의성을 도모하기 위한 개발 시도가 진행 중이다.In addition, attempts to develop hemoglobin A to replace FVIII's function as a blood coagulation factor through a route other than the existing FVIII, and to promote the sustained effect and convenience of the patient.
그 중 하나는 FVIII-mimetic bi-specific 항체로 이는 FIX(또는 activated FIX)과 FX에 특이적인 2종류의 Fab를 포함하는 온전한 IgG 형태의 이특이적 항체로 체내에서 FVIII의 기능을 대신하도록 고안된 것이다 (Kitazawa et al, Nature medicine 2012 18 (10): 1570-4.).One is the FVIII-mimetic bi-specific antibody, which is an intact IgG form of bispecific antibody containing two types of Fab specific to FIX (or activated FIX) and FX, designed to replace the function of FVIII in the body ( Kitazawa et al, Nature medicine 2012 18 (10): 1570-4.).
다른 하나는 항-TFPI(Tissue factor pathway inhibitor)의 사용이다. 이는 TFPI의 기능을 억제하는 물질로 항체, 펩타이드 이외에 앱타머의 형태로도 개발 중이다 (Hilden et al., BLOOD 2012, 119 (24): 5871-8.). TFPI는 TF(tissue factor)-activated FVII 복합체가 FX를 활성화하는 과정을 방해하여 FXa의 생성을 저하하고 FXa에 의해 트롬빈이 생성되지 않도록 하여 혈액응고의 진행을 정지시키는 기능을 한다.The other is the use of anti-issue factor pathway inhibitors (TFPI). It is a substance that inhibits the function of TFPI and is being developed in the form of aptamers in addition to antibodies and peptides (Hilden et al., BLOOD 2012, 119 (24): 5871-8.). TFPI functions to stop the progression of blood coagulation by interfering with the process of the TF (activated FVII) -activated FVII complex, lowering the production of FXa and preventing thrombin from being produced by FXa.
또 다른 하나는 Anti-thrombin 억제제로 이는 혈중 트롬빈 억제제의 기능을 저해함으로써 트롬빈의 활성을 향상시켜 혈우병 동물 또는 혈우병 환자의 혈액 응고가 용이하게 하는 방식으로 작용한다.Another is an anti-thrombin inhibitor, which acts in a way that enhances thrombin activity by inhibiting the function of the thrombin inhibitor in the blood, thereby facilitating blood coagulation in hemophilia animals or hemophilia patients.
그러나 이와 같은 접근법은 혈우병 A형 또는 B형에서 부족되는 FVIII와 FIX 단백질의 활성을 직접 대체하거나 보강하는 것이 아닌 간접적인 방법으로 FVIII의 활성을 모사하거나 우회하는 것으로 치료 기전이 복잡하여 임상에서 안전성과 효력이 충분히 입증되어야 한다. However, this approach is an indirect method that mimics or bypasses the activity of FVIII, rather than directly replacing or enhancing the activities of FVIII and FIX proteins that are lacking in hemophilia type A or B. Effectiveness must be fully demonstrated.
대한민국 공개특허 공보 2014-0114266는 증가된 특이 활성도를 갖는 항혈우병 인자 VIII을 개시하고 있다. Korean Unexamined Patent Publication No. 2014-0114266 discloses anti-hemopathy factor VIII with increased specific activity.
현재까지 개발된 치료제는 기능적 측면에서 투여 주기(정맥 주사)가 짧고, 또한 짧은 반감기로 인한 체내 효력 소실에 따라 잦은 출혈 빈도가 문제점으로 지적되고 있다. 또한 생산성 측면에서 낮은 생산성과 고가의 치료비도 해결되어야 할 문제이다. 따라서 향상된 반감기를 갖으면서도 높은 생산성을 갖는 FVIII 인자의 개발이 필요하다.The therapeutic agents developed to date have been pointed out as a problem of frequent bleeding due to short functional cycle (intravenous injection) and functional loss due to short half-life. In addition, in terms of productivity, low productivity and expensive treatment costs must be solved. Therefore, there is a need for the development of a high productivity FVIII factor with improved half-life.
본원은 체내 체류 시간이 향상되고 양산이 용이한 단쇄 FVIII을 제공하고자 한다. The present application seeks to provide short-chain FVIII with improved residence time in the body and easy mass production.
한 양태에서 본원은 인간 혈액응고 8인자(Factor VIII)의 중쇄, 경쇄 및 일부가 결실된 B 영역 단편을 포함하며, 상기 일부가 결실된 B 영역 단편은 퓨린 단백질분해효소에 의한 절단부위를 포함하지 않도록 서열번호 1의 서열의 1648 및 1649 잔기를 포함하여 상기 잔기의 N-말단 및 C-말단 방향으로 각각 최소 5개의 아미노산이 결실되고, 4개 내지 6개의 당화 부위를 포함하도록 일부가 결실된 것인, 단쇄 혈액응고 8인자(Factor VIII)를 제공한다. In one embodiment the present disclosure includes a heavy region, a light chain and a portion of a B region fragment deleted from human coagulation factor 8 (Factor VIII), wherein the portion of the B region fragment lacking a cleavage site by purine protease. At least 5 amino acids each in the N-terminal and C-terminal directions of the residue, including 1648 and 1649 residues of SEQ ID NO: 1, and partly deleted to include 4 to 6 glycosylation sites Phosphorus, short-chain clotting factor 8 (Factor VIII) is provided.
본원의 일 구현예에서 따르면 본원의 단쇄 혈액응고 8인자에 포함되는 B 영역 단편은 야생형 B domain 서열의 약 15% 내지 40%를 포함하는 것이나, 본원에 따른 효과를 달성하는 한 이로 제한하는 것은 아니다. 당업자라면, 상술한 단쇄 혈액응고 8인자에 포함되는 B 영역 단편의 조건을 고려하여 적절한 단쇄 혈액응고 8인자를 선택할 수 있다. 본원에서 B 영역은 후술하는 바와 같이 a3 영역을 포함하는 것으로 해석된다.According to an embodiment of the present disclosure, the B region fragment included in the single-chain coagulation factor 8 of the present disclosure includes about 15% to 40% of the wild-type B domain sequence, but is not limited thereto as long as the effect according to the present disclosure is achieved. . Those skilled in the art can select an appropriate single-chain coagulation factor 8 in consideration of the conditions of the B region fragment included in the above-mentioned short-chain coagulation factor 8 described above. In the present application, the B region is interpreted to include an a3 region as described later.
본원의 일 구현예 따르면 상술한 조건을 만족하는 일부가 결실된 B 영역 단편에서 결실된 부분은 연속적 또는 비연속적일 수 있으며, 서열번호 1의 서열을 기준으로 상기 일부가 결실된 B 영역 단편은 서열번호 1의 서열을 기준으로 (i) 아미노산 잔기 741 내지 902 및 1654 내지 1689; (ii) 아미노산 잔기 741 내지 965 및 1654 내지 1689의 아미노산 서열; 또는 (iii) 아미노산 잔기 741 내지 902, 1637 내지 1642 및 1654 내지 1689;의 서열로 표시될 수 있다. According to an embodiment of the present disclosure, a portion deleted from a region B deleted from a portion that satisfies the above condition may be continuous or discontinuous, and the region B deleted from the region based on the sequence of SEQ ID NO. Based on the sequence of No. 1 (i) amino acid residues 741 to 902 and 1654 to 1689; (ii) amino acid sequences of amino acid residues 741 to 965 and 1654 to 1689; Or (iii) amino acid residues 741-902, 1637-1642 and 1654-1689.
본원의 일 구현예에 따르면, 본원의 단쇄 혈액응고 8인자는 서열번호 2 내지 7로 표시되는 아미노산 서열 또는 이와 약 90% 이상 상동성이 있는 서열을 갖는 것을 포함하는 것으로, 후술하는 상동성 결정 방법 및 본원에 따른 인자와 생물학적 기능 균등성에 대한 개시내용을 참조하여 적절하게 결정할 수 있을 것이다.According to an embodiment of the present disclosure, the single-chain coagulation factor 8 of the present application includes an amino acid sequence represented by SEQ ID NO: 2 to 7 or a sequence having about 90% or more homology thereto, and the method of determining homology described below. And the disclosure of factors and biological function equality according to the present disclosure may be appropriately determined.
본원에 따른 일 구현예에서 서열번호 2 내지 7로 표시되는 아미노산 서열을 갖는 본원의 단쇄 혈액응고 8인자를 코딩하는 핵산분자는 서열번호 10 내지 15로 표시되거나, 또한 상기 핵산 서열이 세포에서의 발현을 위해 코돈 최적화된 서열로 치환된 것 또는 degenerate codon으로 인한 핵산 서열이 변이된 것을 또한 포함한다.In one embodiment according to the present application, the nucleic acid molecule encoding the single-chain coagulation factor 8 having the amino acid sequence represented by SEQ ID NOs: 2 to 7 is represented by SEQ ID NOs: 10 to 15, or the nucleic acid sequence is expressed in a cell. Also included are those substituted with codon optimized sequences or variants with nucleic acid sequences due to degenerate codons.
본원에 따른 단쇄 혈액응고 8인자는 CS 또는 APTT 방법으로 측정한 비활성이 이쇄 혈액응고 8인자 비활성의 90% 이상이다.The single-chain coagulation factor 8 according to the present invention is 90% or more of the double-chain coagulation factor inactivation measured by the CS or APTT method.
다른 양태에서 본원은 친수성 폴리머, 예를 들면 폴리에틸렌글리콜(Polyethylene glycol), 폴리에틸렌 옥사이드, 덱스트란 또는 폴리시알릭을 포함하는 폴리머와 접합되고, PEG가 사용되는 경우에는 접합 위치에서 acryloyl, sulfone 또는 maleimide를 통하여 접합 될 수 있다. In another embodiment the present invention is conjugated with a hydrophilic polymer, such as a polymer comprising polyethylene glycol, polyethylene oxide, dextran or polysialic, and when PEG is used, it may be via acryloyl, sulfone or maleimide at the conjugation position. Can be spliced.
본원의 일 구현예에 따르면 접합된 혈액응고 8인자는 상기 A 영역 및/또는 B 영역 단편의 일부 아미노산 잔기에서 친수성 폴리머와 컨쥬게이션되고, 상기 컨쥬게이션되는 위치는 상기 B 영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 754, 781, 782, 788, 789, 825 및 897로 구성되는 군으로부터 선택되는 하나 이상의 위치이고; 상기 A-영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 491, 495, 498 및 1806으로 구성되는 군으로부터 선택되는 하나 이상의 위치이고, 상기 컨쥬게이션되는 위치의 잔기는 상기 친수성 폴리머와의 접합을 위해 시스테인으로 치환될 수 있다. According to an embodiment of the present disclosure, the conjugated coagulation factor 8 is conjugated with a hydrophilic polymer at some amino acid residues of the A region and / or the B region fragment, and the conjugated position is the sequence of SEQ ID NO: 1 in the B region. At least one position selected from the group consisting of amino acid residues 754, 781, 782, 788, 789, 825 and 897; At least one position selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1 in the A-region, wherein the residue at the conjugated position is for conjugation with the hydrophilic polymer It may be substituted with cysteine.
본원에 따른 일 구현예에서는 PEG와 접합되며, 특히 평균 분자량이 20kDa 이상의 PEG가 사용된다. In one embodiment according to the invention conjugated with PEG, in particular PEG with an average molecular weight of at least 20 kDa is used.
다른 양태에서 본원은 또한 본원에 따른 단쇄 혈액응고 8인자, 특히 서열번호 2 내지 7로 표시되는 단쇄 혈액응고 8인자, 또는 상기 아미노산 서열에서 친수성 폴리머와의 접합을 위해 상기 B 영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 754, 781, 782, 788, 789, 825 및 897로 구성되는 군으로부터 선택되는 하나 이상의 위치이고; 상기 A-영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 491, 495, 498 및 1806으로 구성되는 군으로부터 선택되는 하나 이상의 위치의 해당 아미노산 잔기가 시스테인으로 치환된 단쇄 혈액응고 8인자, 또는 치환된 시스테인기를 포함하는 단쇄 혈액응고 8인자를 코딩하는 핵산분자를 제공한다. In another embodiment the application is also directed to the short chain hemagglutination factor 8 according to the invention, in particular to the short chain hemagglutination factor 8 represented by SEQ ID NOS: 2 to 7, or to the region B of the sequence B for conjugation with a hydrophilic polymer in said amino acid sequence. One or more positions selected from the group consisting of amino acid residues 754, 781, 782, 788, 789, 825, and 897, based on the sequence; A single-chain hemagglutination factor 8 substituted with cysteine at one or more positions corresponding to at least one position selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1 in the A-region A nucleic acid molecule encoding a single-chain clotting factor 8 containing a cysteine group is provided.
본원에 따른 일 구현예에서 시스테인이 도입된 잔기는 표 3에 기재된 바와 같고, 친수성 폴리머와의 접합을 위해 해당 잔기가 시스테인으로 치환된 핵산분자는 서열번호 17 내지 32로 표시되며, 이에 상응하는 아미노산 서열은 서열번호 33 내지 48로 표시된다. 또한 상기 핵산 서열이 세포에서의 발현을 위해 코돈 최적화된 서열로 치환된 것 또는 degenerate codon으로 인한 핵산 서열이 변이된 것을 또한 포함한다. In one embodiment according to the present invention, the residues into which cysteine is introduced are shown in Table 3, and the nucleic acid molecules whose residues are substituted with cysteine for conjugation with a hydrophilic polymer are represented by SEQ ID NOs: 17 to 32, and corresponding amino acids. The sequence is represented by SEQ ID NOs: 33-48. Also included are those in which the nucleic acid sequence has been replaced with a codon optimized sequence for expression in a cell or the nucleic acid sequence due to degenerate codons is mutated.
다른 양태에서 본원은 또한 본원에 따른 핵산분자를 포함하는 벡터, 상기 벡터를 포함하는 세포를 제공한다. In another aspect the present disclosure also provides a vector comprising a nucleic acid molecule according to the present application, a cell comprising the vector.
또 다른 양태에서 본원은 또한 본원에 따른 벡터 또는 세포를 이용한 단쇄 혈액응고 8인자의 생산방법을 제공하며, 일 구현예에 따르면 본원에 따른 방법은 본원에 따른 벡터를 진핵세포에 전달이입하는 단계, 또는 선택적으로 본원에 따른 벡터가 전달된 세포를 제공하는 단계: 상기 세포를 배양액 중에서 배양을 통하여 도입된 시스테인이 자유 티올 기를 시스테인이나 글루타티온에 의해 이황화(disulfide) 결합으로 마스킹 된 단쇄 FVIII 형태로 발현시키는 단계; 상기 배양액으로부터 발현된 단쇄 FVIII을 수집하여 환원제로 처리하여 마스킹 된 시스테인이나 글루타티온을 이탈시키는 단계; 및 상기 처리된 배양액을 페길화완충액으로 처리하는 단계를 포함한다. In another aspect the present invention also provides a method of producing a single-chain blood coagulation factor 8 using a vector or a cell according to the present invention, according to one embodiment the method according to the present invention comprises the steps of transferring the vector according to the present application to eukaryotic cells, Or optionally providing a cell to which a vector according to the present invention has been delivered: expressing the cells in a single chain FVIII form in which the cysteine introduced through the culture in the culture medium is free thiol group masked by disulfide bonds by cysteine or glutathione. step; Collecting short-chain FVIII expressed from the culture solution and treating it with a reducing agent to release masked cysteine or glutathione; And treating the treated culture solution with PEGylated buffer solution.
다른 양태에서 본원은 또한 청구범위를 포함하는 본원에 개시된 어느 하나의 단쇄 혈액응고 8인자를 포함하는 혈우병 환자 또는 혈액의 응고가 필요한 환자에 사용하기 위한 혈액 응고 키트, 상기 단쇄 혈액응고 8인자의 혈우병 치료용 용도 또는 조성물, 또는 단쇄 혈액응고 8인자를 혈우병 환자 또는 혈액의 응고가 필요한 환자에게 치료적으로 유효한 양으로 투여하는 단계를 포함하는 혈액응고 방법을 제공한다.In another aspect the present invention also provides a blood coagulation kit for use in a hemophilia patient or any patient in need of blood coagulation comprising any of the short chain coagulation 8 factors disclosed herein, including the claims, hemophilia of the short chain coagulation factor 8 A therapeutic coagulation method comprising administering a therapeutic use or composition, or a short-chain coagulation factor 8, to a hemophilia patient or a patient in need of coagulation in a therapeutically effective amount.
본원에 따른 단쇄 FVIII는 경쇄 및 중쇄가, 일부 서열이 결실된 B-도메인으로 연결되며, 특히 정상적인 FVIII 발현 과정 중에 발생하는 furin 단백질분해효소에 의해 절단되는 절단부위를 포함하지 않으며, 최소 4개의 당화 부위를 포함하도록 일부가 결실된 B 영역을 포함한다. 본원에 따른 단쇄 혈액응고 8인자(Factor VIII) 또는 상기 A 또는 B 영역의 일부 잔기가 페길화된 단쇄 혈액응고 8인자로서 활성은 그대로 유지하면서 단쇄의 형태로 제조되어 양산이 용이할 뿐 아니라 페길화를 통해 체내 반감기가 증가하여 혈우병 A형의 치료제로서 환자의 편리성을 증대시켰음은 물론 생산비용의 절감을 통해 치료비의 절감을 가져올 수 있다.Single-chain FVIII according to the present invention is a light chain and heavy chain, which is linked to the B-domain lacking some sequences, in particular does not include a cleavage site cleaved by furin protease that occurs during normal FVIII expression process, at least 4 It includes the B region partially deleted to include the site. The short-chain coagulation factor 8 (Factor VIII) or some residues of the A or B region according to the present invention are pegylated short-chain coagulation factor 8, which are prepared in the form of short chains while maintaining the activity thereof, and thus easy to mass-produce. Through the increase in the half-life of the body through the treatment of hemophilia type A, as well as increased the convenience of the patient can reduce the cost of treatment through the reduction of production costs.
도 1은 본원의 일 구현예에 따른 페길화된 단쇄 FVIII 구조를 도식적으로 나타낸 것으로, 1648 잔기는 furin의 절단위치를 나타낸다.Figure 1 schematically shows a PEGylated short-chain FVIII structure according to an embodiment of the present application, 1648 residues indicate the cleavage position of furin.
도 2는 본원의 일 구현예에 따른 전장 FVIII으로부터 단쇄 FVIII 제작을 도식적으로 나타낸 것이다. Furin 절단부위(서열 1 기준으로 1648 번째 아미노산 서열)를 표시하였으며 도면에 기재된 숫자는 표시된 부위에 상응하는 각 영역을 아미노산 잔기로 나타낸 것이다.Figure 2 schematically shows the production of short chain FVIII from full length FVIII according to one embodiment of the present application. Furin cleavage sites (the 1648 th amino acid sequence based on SEQ ID NO: 1) are indicated and the numbers in the figures represent each region corresponding to the indicated sites as amino acid residues.
도 3은 본원의 일 구현예에 따른 단쇄 FVIII 발현을 웨스턴블랏으로 분석한 결과이다.Figure 3 is a result of Western blot analysis of single-chain FVIII expression according to an embodiment of the present application.
도 4는 본원의 일 구현예에 따른 최적의 단쇄 FVIII의 페길화 위치 선정을 위해 테스트한 페길화 위치를 표시한 것이다.4 shows the PEGylation position tested for optimal PEGylation position of short chain FVIII according to one embodiment of the present disclosure.
도 5는 본원의 일 구현예에 따른 자유 시스테인이 도입된 변이형 단쇄 FVIII 발현 후 발현된 FVIII 활성을 분석한 결과이다.5 is a result of analyzing the FVIII activity expressed after the expression of the variant single-chain FVIII introduced with free cysteine according to an embodiment of the present application.
도 6은 본원의 일 구현예에 따른 자유 시스테인이 도입된 변이형 단쇄 FVIII의 페길화 후 SDS-PAGE 분석 결과이며, 별표로 표시된 라인은 페길화 효율이 상대적으로 높은 것을 나타낸다. 6 is a result of SDS-PAGE analysis after PEGylation of the mutant short-chain FVIII in which free cysteine is introduced according to an embodiment of the present application, and the lines marked with asterisks indicate that the PEGylation efficiency is relatively high.
도 7은 본원의 일 구현예에 따른 scFVIII의 PK(pharmacokinetics)를 상용화 FVIII 물질과 비교한 시험결과이다. 7 is a test result comparing the pharmacokinetics (PK) of scFVIII with a commercially available FVIII material according to an embodiment of the present application.
도 8a 및 8b는 각각 본원의 일 구현예에 따른 페길화 scFVIII의 tail bleeding 지혈 효력 시험을 상용화 FVIII 물질의 효능과 비교하기 위한 실험 방법의 개략도, 및 실험결과이다. 도 8b에서 PEG40kDa-scFVIII-B3를 나타낸다.8A and 8B are schematic diagrams and experimental results for comparing the tail bleeding hemostatic efficacy test of PEGylated scFVIII with the efficacy of commercialized FVIII material, respectively, according to one embodiment of the present application. In FIG. 8B PEG40kDa-scFVIII-B3 is shown.
본원은 생산성과 체내 안정성이 증가된 단쇄형태로 발현될 수 있는 인간 혈액응고 8인자(Coagulation Factor VIII) 변이체의 개발 그리고 이러한 변이체의 페길화를 통하여 환자의 투여 주기의 편의성을 제고시킨 혈액응고 인자의 개발을 근거로 한다. The present invention describes the development of a coagulation factor VIII variant that can be expressed in a short chain form with increased productivity and stability in the body, and the coagulation factor of the coagulation factor that improves the convenience of the patient's administration cycle through PEGylation of such a variant. Based on development.
혈액응고 8인자는 A1-A2-B-A3-C1-C2 영역으로 구성되며, 간세포에서 단쇄의 단백질로 합성된다. 합성 후 프로세싱을 거쳐 성숙되어 중쇄와 경쇄로 구성된 280kDa의 헤테로이량체를 형성한다. 이 중 경쇄는 80kDa이며 A3-C1-C2 영역으로 구성되고, 중쇄는 A1-A2-B 영역으로 구성되고 분자량은 90-200kDa으로 B 영역의 길이와 당화 정도의 차이로 인해 분자량의 차이가 크다. A1 domain과 A2 domain 사이의 a1 domain 및 A2 domain과 B domain 사이의 a2 domain 그리고 a3 domain은 중쇄에 포함된다. 이러한 헤테로이량체는 혈액 내에서 vWF와 결합한 비활성화 상태로 존재하다가, 혈관 손상과 같은 자극에 노출시, 트롬빈에 의해 알지닌 372, 740 및 1689번 잔기 뒤에서 절단된다. 그 결과 vWF로부터 분리되어, 활성화되어 A1, A2 및 A3-C1-C2의 삼량체가 형성된다. 이어 삼량체는 FIXa에 의한 FX의 활성화를 촉매하고, 빠르게 비활성화된다. Coagulation factor 8 consists of the A1-A2-B-A3-C1-C2 regions and is synthesized as a short-chain protein in hepatocytes. After synthesis, it is processed and matured to form 280kDa heterodimer consisting of heavy and light chains. Among them, the light chain is 80kDa and is composed of A3-C1-C2 region, the heavy chain is composed of A1-A2-B region and the molecular weight is 90-200kDa, which has a large molecular weight difference due to the difference in the length and degree of glycation of the B region. The a1 domain between the A1 and A2 domains and the a2 and a3 domains between the A2 and B domains are included in the heavy chain. This heterodimer is present in the blood in an inactive state associated with vWF, and is cleaved by thrombin behind residues 372, 740 and 1689 upon exposure to stimuli such as vascular damage. As a result, it is separated from vWF and activated to form a trimer of A1, A2 and A3-C1-C2. The trimer then catalyzes the activation of FX by FIXa and is rapidly deactivated.
단쇄 FVIII는 구조적으로 안정하고, 발현량이 증가하는 것으로 보고되어 있다 (WO2004/067566 참조). 이쇄 형태의 FVIII에서는 B 영역의 길이에 따른 발현량 차이가 있음이 보고되었다 (Miao et al., Blood. 2004 May 1;103(9):3412-9.). Short-chain FVIII is reported to be structurally stable and to increase expression (see WO2004 / 067566). In the double-chain type FVIII, there was reported a difference in expression according to the length of the B region (Miao et al ., Blood. 2004 May 1; 103 (9): 3412-9.).
본원에서는 단쇄 FVIII의 제작에 있어서 a3를 포함하는 B 영역의 길이와 서열에 따른 발현량 차이 이외에, 특이적 활성도(specific activity)와 APTT/CS 비에도 변화가 초래됨을 확인하였으며, B 영역의 길이 및 서열을 최적으로 조절하여 특이적 활성도와 APTT/CS 비가 이쇄의 천연형과 유사한 재조합 단쇄 FVIII을 제조하였다. 본원에 따른 단쇄 FVIII은 천연형 FVIII 고유의 특성을 유지하면서도 안정하고 발현량이 우수한 것으로 나타났다.In the present application, it was confirmed that in addition to the length of the B region including a3 and the expression amount according to the sequence in the production of the single-chain FVIII, a change was caused in the specific activity and the APTT / CS ratio. The sequence was optimally adjusted to produce recombinant single chain FVIII with specific activity and APTT / CS ratio similar to the native of the double chain. Short-chain FVIII according to the present application was found to be stable and excellent in expression while maintaining the inherent properties of native FVIII.
따라서 한 양태에서 본원은 인간 혈액응고 8인자(Factor VIII)의 중쇄 및 경쇄 부위가, 일부 서열이 결실된 B- 도메인으로 연결된 형태의 단쇄 FVIII으로, 특히 상기 일부 서열이 결실된 B- 도메인은 단백질분해효소(furin)에 의한 절단부위를 포함하지 않도록 서열번호 1의 1648 및 1649 잔기 위치를 기준으로 이를 포함하여 각각 N-말단 및 C-말단 방향으로 각각 최소 5개의 아미노산이 결실되고, 4개 내지 6개의 당화 부위를 포함하도록 일부가 결실된 B 영역 단편을 포함한다. Thus, in one embodiment, the present application is a single-chain FVIII in which the heavy and light chain regions of human coagulation factor (Factor VIII) are linked by a B-domain in which some sequences are deleted, in particular the B-domain in which the partial sequences are deleted is a protein. At least 5 amino acids are respectively deleted in the N-terminal and C-terminal directions, respectively, based on the 1648 and 1649 residue positions of SEQ ID NO: 1 so as not to include a cleavage site by a furin, and 4 to And a B region fragment partially deleted to contain six glycosylation sites.
본원에 따른 단쇄 FVIII은 A1- A2- B 일부- A3- C1- C2 영역을 포함한다. 본원에 포함된 각 영역의 FVIII에서의 잔기 위치 및 그 서열은 공지된 것으로 인간 아미노산 서열 예를 들면 인간 FVIII은 서열번호 1의 서열로 표시될 수 있으며, 이를 기준으로 중쇄는 A1 및 A2 영역을 포함하는 1-740 잔기, B 영역은 741-1689 잔기, A3-, C1, 및 -C2 영역을 포함하는 경쇄는 1690-2332 잔기까지이다 (도 2; 및 Orlova et al., Acta Naturae. 2013 Apr-Jun; 5(2): 19-39. 참조). 본원에서 B 영역은 도 2에 기재된 a3 영역(1649 내지 1689)을 포함하여 언급하는 것이다. Single chain FVIII according to the present invention comprises an A1-A2-B partially-A3-C1-C2 region. The residue positions and the sequences in FVIII of each region included herein are known and human amino acid sequences such as human FVIII can be represented by the sequence of SEQ ID NO: 1 with heavy chains comprising the A1 and A2 regions. 1-740 residues, the B region is 741-1689 residues, the light chain comprising the A3-, C1, and -C2 region is up to 1690-2332 residues (Fig. 2; and Orlova et al ., Acta Naturae. 2013 Apr- Jun; 5 (2): 19-39. Region B is referred to herein, including the a3 regions 1649-1689 described in FIG. 2.
이런 측면에서 본원에 따른 단쇄 FVIII는 A1-A2-B'-A3-C1-C2의 식으로 표시될 수 있으며, A1은 상기 A1 영역, A2는 A2 영역, B'은 B 영역의 일부, A3는 A3 영역, C1은 C1 영역, C2는 C2 영역을 의미하며, 상기 각 영역은 앞서 언급한 바를 참조할 수 있다. In this respect, short-chain FVIII according to the present application may be represented by the formula A1-A2-B'-A3-C1-C2, A1 is the A1 region, A2 is the A2 region, B 'is part of the B region, A3 is A3 region, C1 means C1 region, C2 means C2 region, each of which can be referred to above.
본원에 따른 단쇄 FVIII에 포함되는 일부가 결실된 B 영역(B')은 단백질분해효소 furin 절단부위가 결실된 것으로 일 구현예에서는 서열번호 1의 1648 및 1649 잔기를 포함하며 상기 각 잔기의 N-말단 및 C-말단 각 방향으로 최소 5개의 아미노산이 결실된 것이 포함된다. B 영역에는 다수의 당화 부위가 존재하며, 단백질의 당화는 단백질의 활성과 발현 과정 중 세포 내부 및 발현 후 세포 외부에서 안정성에도 영향을 미치기 때문에, 상기 B 영역의 결실부위를 결정함에 있어서 포함되는 당화 개수를 고려할 수 있다. The B region (B ′) which is partially deleted in the short chain FVIII according to the present invention is deleted from the protease furin cleavage site and in one embodiment includes 1648 and 1649 residues of SEQ ID NO: 1 and Deletions of at least five amino acids in each of the terminal and C-terminal directions. There are a number of glycosylation sites in the B region, and glycosylation of the protein affects the activity of the protein and its stability in the cell during and after expression and also outside the cell. The number can be considered.
본원에 따른 일 구현예에서는 특히 당화 개수 4개 내지 6개가 포함되도록 B 영역의 결실부위가 결정된다. In one embodiment according to the present invention, the deletion region of the B region is determined to include 4 to 6 glycosylation.
또한 B 영역의 결실부위를 결정함에 있어서 본원에 따른 단쇄 FVIII에 포함되는 furin 절단부위가 결실된 B 영역은 전체 야생형 B-domain의 약 15~40%를 포함하는 한도에서 결정될 수 있다. In addition, in determining the deletion region of the B region, the B region in which the furin cleavage region included in the short chain FVIII according to the present invention is deleted may be determined in a limit including about 15 to 40% of the total wild type B-domain.
또한 본원에 따른 단쇄 FVIII에 포함되는 일부가 결실된 B 영역의 결실 부위는 연속적 또는 비연속적일 수 있다. In addition, the deletion site of the B region in which a part included in the short chain FVIII according to the present invention is deleted may be continuous or discontinuous.
일 구현예에서 본원에 따른 단쇄 FVIII에 포함되는 furin 절단부위를 포함하는 연속적으로 결실된 B 영역을 포함하는 경우는 서열번호 1의 서열을 기준으로 (i) 아미노산 잔기 741 내지 902 및 1654 내지 1689 (즉 B 영역의 903 내지 1653 잔기가 연속적으로 결실됨); (ii) 아미노산 잔기 741 내지 965 및 1654 내지 1689 (즉, B 영역의 966 내지 1653 잔기가 결실됨)로 표시되는 서열이거나, 또는 비연속적으로 결실된 B 영역을 포함하는 경우는 (iii) 아미노산 잔기 741 내지 902, 1637 내지 1642 및 1654 내지 1689의 서열로 표시된다.In one embodiment, when comprising a continuously deleted B region comprising a furin cleavage site included in the single-chain FVIII according to the present application based on the sequence of SEQ ID NO: 1 (i) amino acid residues 741 to 902 and 1654 to 1689 ( Ie 903 to 1653 residues in the B region are deleted in succession); (ii) a sequence represented by amino acid residues 741 to 965 and 1654 to 1689 (ie, the 966 to 1653 residues in the B region are deleted), or when it comprises a noncontiguously deleted B region (iii) 741 to 902, 1637 to 1642 and 1654 to 1689.
다른 일 구현예에서 상기와 같은 결실된 B 영역을 포함하는, 본원에 따른 FVIII 아미노산 서열은 서열번호 2 내지 7로 표시될 수 있다.In another embodiment, the FVIII amino acid sequence according to the present disclosure, including the deleted B region, may be represented by SEQ ID NOs: 2 to 7.
본원 개시된 재조합 FVIII은 위와 같은 서열로 한정되는 것이 아니며, 이의 생물학적 균등물(biologically equivalents)을 포함하는 것이다. 생물학적 균등물이란, 본원에 개시된 아미노산 서열에 추가적인 변형을 가하였으나, 본원에 따른 폴리펩타이드와 실질적으로 동일한 활성을 갖는 것으로, 이러한 변형은, 예를 들어 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함하는 것이다.The recombinant FVIII disclosed herein is not limited to such sequences, but includes biologically equivalents thereof. Biological equivalents are those which have additional modifications to the amino acid sequences disclosed herein, but which have substantially the same activity as the polypeptides according to the invention, such modifications may, for example, delete, insert and / or substitute amino acid sequence residues. It is to include.
일 구현예에서는 본원에 따른 재조합 FVIII 폴리펩타이드에 보존적 아미노산 치환이 일어난 것을 포함한다.In one embodiment, conservative amino acid substitutions have occurred in a recombinant FVIII polypeptide according to the present disclosure.
보존적 아미노산 치환(conservative amino acid substitution)이란 특정 폴리펩타이드가 갖는 활성을 실질적으로 영향을 미치거나 감소시키지 않는 치환을 의미하는 것으로, 예를 들면, 1 내지 15개의 보존적 치환, 1 내지 12개, 예를 들면 1, 2, 5, 7, 9, 12, 또는 15개의 보존적 아미노산 치환을 포함할 수 있다.Conservative amino acid substitutions mean substitutions that do not substantially affect or reduce the activity of a particular polypeptide. For example, 1 to 15 conservative substitutions, 1 to 12, For example, 1, 2, 5, 7, 9, 12, or 15 conservative amino acid substitutions.
보존적 아미노산 치환은 당업계에 알려진 것으로 예를 들면 Blosum(BLOcks SUbstitution Matrix) 기반한 Creighton (1984) Proteins. W. H. Freeman and Company (Eds)의 기재된 사항; 및 Henikoff, S.; Henikoff, J.G. (1992). "Amino Acid Substitution Matrices from Protein Blocks". PNAS 89 (22): 10915-10919. doi:10.1073/pnas.89.22.10915; WO2009012175 A1 등에 기재된 것을 참조할 수 있다.Conservative amino acid substitutions are known in the art, for example Creighton (1984) Proteins, based on Blosum (BLOcks SUbstitution Matrix). As described in W. H. Freeman and Company (Eds); And Henikoff, S .; Henikoff, J.G. (1992). "Amino Acid Substitution Matrices from Protein Blocks". PNAS 89 (22): 10915-10919. doi: 10.1073 / pnas.89.22.10915; Reference may be made to WO2009012175 A1 and the like.
이러한 아미노산 변이 특히 치환은 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 갖으며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다. 따라서, 이러한 것을 고려하면, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.Such amino acid variations, in particular substitutions, are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have a similar shape. Thus, considering this, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.
또한 아미노산 치환을 도입하는 데 있어서, 아미노산의 소수성 인덱스(hydropathic index)가 고려될 수 있다. 각각의 아미노산은 소수성과 전하에 따라 소수성 인덱스가 부여되어 있다: 아이소루이신(+4.5); 발린(+4.2); 루이신(+3.8); 페닐알라닌(+2.8); 시스테인/시스타인(+2.5); 메티오닌(+1.9); 알라닌(+1.8); 글라이신(-0.4); 쓰레오닌(-0.7); 세린(-0.8); 트립토판(-0.9); 타이로신(-1.3); 프롤린(-1.6); 히스티딘(-3.2); 글루타메이트(-3.5); 글루타민(-3.5); 아스파르테이트(-3.5); 아스파라긴(-3.5); 라이신(-3.9); 및 아르기닌(-4.5). 상기와 같은 소수성 인덱스는 특히 단백질의 상호적인 생물학적 기능(interactive biological function)을 부여하는 데 있어서 중요하다. 유사한 소수성 인덱스를 가지는 아미노산으로 치환하여야 유사한 생물학적 활성을 보유할 수 있다는 것은 공지된 사실이다. 소수성 인덱스를 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 ±1 이내, 보다 더 바람직하게는 ±0.5 이내의 소수성 인덱스 차이를 나타내는 아미노산 간의 치환이 유리하다.In addition, in introducing amino acid substitutions, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5). Such hydrophobicity indexes are particularly important for imparting the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5 are advantageous.
또한 유사한 친수성 수치(hydrophilicity value)를 가지는 아미노산 사이의 치환이 균등한 생물학적 활성을 갖는 단백질을 초래한다는 것도 잘 알려져 있다. 예를 들면 미국 특허 제4,554,101호에 개시된 바와 같이, 다음의 친수성 값이 각각의 아미노산 잔기에 부여되어 있다: 아르기닌(+3.0); 라이신(+3.0); 아스팔테이트(+3.0±1); 글루타메이트(+3.0±1); 세린(+0.3); 아스파라긴(+0.2); 글루타민(+0.2); 글라이신(0); 쓰레오닌(-0.4); 프롤린(-0.5±1); 알라닌(-0.5); 히스티딘(-0.5); 시스테인(-1.0); 메티오닌(-1.3); 발린(-1.5); 루이신(-1.8); 아이소루이신(-1.8); 타이로신(-2.3); 페닐알라닌(-2.5); 트립토판(-3.4). It is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed, for example, in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).
또한 본원에 따른 재조합 단백질의 활성을 전체적으로 변경시키지 않는 아미노산 치환은 당해 분야에 공지되어 있다 (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 예를 들면 가장 통상적으로 일어나는 치환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu 및 Asp/Gly 간의 치환을 들 수 있다. Amino acid substitutions that do not alter the activity of the recombinant protein according to the present invention are also known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). For example the most commonly occurring substitutions are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe And substitutions between Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.
따라서, 상술한 바와 같은 생물학적 균등성을 갖는 변이체는 본원에 개시된 아미노산 서열 또는 이를 코딩하는 핵산분자와 실질적 동일성을 갖는 것으로 본원의 범위에 포함된다.Thus, variants having biological equivalents as described above are included within the scope of this application as having substantially the same identity as the amino acid sequences disclosed herein or nucleic acid molecules encoding them.
이러한 실질적 동일성은 당업계의 공지된 서열비교 방법을 이용하여 결정될 수 있다. 본원에 개시된 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80% 상동성, 특히 85% 이상, 더욱 특히 90%, 더더욱 특히 95% 이상의 상동성을 나타내는 서열은 실질적 동일성을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. 예를 들면 Smith and Waterman, Adv. Appl. Math. (1981) 2:482; Needleman and Wunsch, J. Mol. Bio. (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al., Nuc. Acids Res. (1988) 16:10881-90; Huang et al., Comp. Appl. BioSci. (1992) 8:155-65 및 Pearson et al., Meth. Mol. Biol. (1994) 24:307-31에 개시되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al., J. Mol. Biol. (1990) 215:403-10)은 NCBI 등에서 접근 가능하며, blast, blastp, blasm, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLAST는 www.ncbi.nlm.nih.gov/BLAST/에서 접속 가능하며, 이 프로그램을 이용한 서열 상동성 비교 방법은 www.ncbi.nlm.nih.gov/BLAST/blast_help.html에서 확인할 수 있다.Such substantial identity can be determined using sequence comparison methods known in the art. At least 80% homology, in particular at least 85%, more when aligning the sequences disclosed herein to any other sequences as closely as possible and analyzing the aligned sequences using algorithms commonly used in the art. In particular, sequences exhibiting at least 90%, even more particularly at least 95% homology, mean substantial identity. Alignment methods for sequence comparison are known in the art. For example, Smith and Waterman, Adv. Appl. Math. (1981) 2: 482; Needleman and Wunsch, J. Mol. Bio. (1970) 48: 443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73: 237-44; Higgins and Sharp, CABIOS (1989) 5: 151-3; Corpet et al., Nuc. Acids Res. (1988) 16: 10881-90; Huang et al., Comp. Appl. BioSci. (1992) 8: 155-65 and Pearson et al., Meth. Mol. Biol. (1994) 24: 307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. (1990) 215: 403-10) is accessible from NCBI and the like, and can be used with blast, blastp, blasm, blastx, tblastn and tblastx. It can be used in conjunction with a sequence analysis program. BLAST can be accessed at www.ncbi.nlm.nih.gov/BLAST/, and sequence homology comparisons using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
본원에 따른 단쇄의 재조합 FVIII(recombinant single chain FVIII)는 활성 물질의 몰당 활성의 측면에서 야생형의 이쇄 FVIII과 비교하여 90% 이상, 95% 이상, 97% 이상, 98% 이상, 99% 이상, 또는 100%를 갖는 것을 포함하는 것이다. 야생형의 이쇄 FVIII과 이의 활성을 측정하는 방법은, 공지되어 있으며, 예를 들면 CS와 clotting assay(APTT: activated partial thromboplastin time)로 측정한 비활성 수치와 (APTT)/CS 비율을 결정하여 수행될 수 있다 (Coagulation Assays Circulation. 2005;112:e53-e60). 당업자라면 본원의 기재, 당업계의 기술 및 참고문헌의 기재를 고려하여 적절한 방법을 선택하여 활성을 측정하고 이에 기반하여 본원의 범위에 포함되는 단쇄 재조합 FVIII를 결정할 수 있을 것이다. 이쇄 FVIII은 상술한 바와 같이 퓨린에 의해 프로세스 되어 경쇄와 중쇄의 이량체로 구성된다. 본원에서는 천연형 FVIII의 재조합 단백질(Advate®)과 비교하였으며 이는 CHO 세포에서 재조합 형태로 발현한 FVIII으로 대부분이 다양한 B 영역의 길이를 포함하는 이쇄로 된 것으로 혈장 유래 천연형의 FVIII의 구성과 유사하다. Recombinant single chain FVIII according to the present disclosure is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or as compared to wild-type double-chain FVIII in terms of activity per mole of active substance. It includes those having 100%. Wild-type double-chain FVIII and methods for measuring its activity are well known and can be performed, for example, by determining the inactivation level and (APTT) / CS ratio measured by CS and the clotting assay (APTT: activated partial thromboplastin time). (Coagulation Assays Circulation. 2005; 112: e53-e60). Those skilled in the art will be able to determine the activity and determine short chain recombinant FVIII included within the scope of the present disclosure based on the disclosure herein, the description in the art, and references by selecting the appropriate method. Bi-chain FVIII is processed by purine as described above to consist of dimers of light and heavy chains. Here, compared to the recombinant protein of native FVIII (Advate®), which is expressed in recombinant form in CHO cells, most of which consist of a double-chain containing various lengths of B region, similar to the composition of plasma-derived native FVIII. Do.
본원에 따른 단쇄 FVIII와 비교대상이 되는 이쇄 FVIII은 B 영역을 포함하거나 포함하지 않는다. B 영역을 포함하지 않는 이쇄의 경우 주로 재조합 방법으로 의도적으로 B 영역을 제거하거나 천연형의 FVIII이 발현되는 과정에서 프로세싱과 혈 중에서 FXa(활성형 FX)와 트롬빈에 의한 단백 절단 프로세스에 의하여 B 영역을 포함하지 않는 이쇄가 생성될 수 있다. B 영역을 포함한 이쇄의 경우 재조합 방법으로 B 영역의 일부 또는 전체를 포함하도록 하거나 천연형의 FVIII이 발현되는 과정에서 프로세스와 혈 중에서 FXa(활성형 FX)와 트롬빈에 의한 단백 절단 프로세스에 의하여 B 영역의 일부 또는 전체를 포함하는 FVIII 이쇄가 생성될 수 있다. 그러나 본원의 단쇄와 직접 비교한 이쇄 FVIII은 B 영역 전체를 포함하는 천연형의 FVIII을 재조합 발현으로 발현한 것으로 세포 내부에서 외부로 배출 시 furin에 의한 프로세싱되어 B 영역 전체를 포함하는 이쇄 FVIII와 그 이후 트롬빈 또는 FIXa 또는 FXa에 의해 B 영역의 일부 또는 전체가 제거되어 B 영역을 전혀 포함하지 않거나 또는 B 영역의 일부를 포함하는 FVIII의 혼합물로 천연형 FVIII와 가장 유사한 재조합 FVIII이다.The double chain FVIII to be compared with the single chain FVIII according to the present application may or may not include the B region. In the case of the double-chain which does not include the B region, the B region is mainly processed by recombination intentionally removing the B region or expressing a native FVIII and processing the protein and cleavage by FXa (active FX) and thrombin in the blood. A backbone that does not contain can be generated. In the case of the double chain including the B region, the B region may be recombinantly included in part or the whole of the B region, or in the process of expressing a native FVIII, or by the protein cleavage process by FXa (active FX) and thrombin in the blood. FVIII duplexes comprising some or all of may be generated. However, the double-chain FVIII, which is directly compared with the short chain of the present application, expresses recombinant FVIII of a native type including the entire B region by recombinant expression. Subsequently, some or all of the B region is removed by thrombin or FIXa or FXa so that it is a recombinant FVIII that is most similar to the native FVIII in a mixture of FVIII containing no B region or a part of the B region.
다른 양태에서 본원은 본원에 따른 단쇄의 재조합 FVIII를 코딩하는 핵산분자 또는 폴리뉴클레오타이드, 상기 폴리뉴클레오타이드를 포함하는 벡터 또는 상기 벡터로 형질전환된 세포(주)에 관한 것이다. In another aspect the present application relates to a nucleic acid molecule or polynucleotide encoding a single-chain recombinant FVIII according to the present application, a vector comprising said polynucleotide or a cell strain transformed with said vector.
본원에 따른 재조합 단쇄 FVIII를 코딩하는 핵산분자는, 본원에 따른 재조합 단쇄 FVIII가 발현되는 세포의 종류에 맞추어 코돈최적화(Codon optimized) 된 것을 포함하는 것이다. 본원에 따른 일 구현예서는 CHO 세포 코돈에 최적화되며, 이러한 핵산 서열은 서열번호 5의 단백질을 코딩하는 핵산 서열을 최적화한 것으로 이는 서열번호 15의 서열로 표시될 수 있다. Nucleic acid molecules encoding the recombinant single-chain FVIII according to the present application includes those that have been codon optimized according to the type of cells expressing the recombinant single-chain FVIII according to the present application. In one embodiment according to the present invention is optimized for the CHO cell codon, the nucleic acid sequence is an optimization of the nucleic acid sequence encoding the protein of SEQ ID NO: 5 can be represented by the sequence of SEQ ID NO: 15.
본원에 따른 재조합 단쇄 FVIII를 코딩하는 핵산분자는 또한 상술한 바와 같이 실질적으로 동일한 생물학적 균등물에 해당하는 FVIII를 코딩하는 것을 또한 포함하는 것이다. Nucleic acid molecules encoding recombinant single-chain FVIII according to the present invention also include those encoding FVIII corresponding to substantially the same biological equivalents as described above.
또한 본원에 따른 단쇄의 재조합 FVIII를 코딩하는 핵산분자는 다양한 목적을 위해 다양한 발현 벡터에 클로닝되어 사용될 수 있다. 발현 벡터의 구체적 구성은 본원에 따른 단쇄 재조합 FVIII을 발현하고자 하는 숙주 세포에 따라 달라질 수 있으나, 본원에 따른 핵산분자의 mRNA로의 발현 또는 mRNA의 단백질로의 발현을 조절하는 서열, 예를 들면 프로모터 및/또는 인핸서 등과 같은 조절서열을 포함한다. 이러한 또는 다양한 다른 목적으로 사용될 수 있는 다양한 벡터 및 조절서열이 공지되어 있으며, 당업자라면 본원의 구체적 목적 및 효과를 고려하여 적절한 것으로 선택할 수 있을 것이며, 예를 들면 본원의 실시예 및 도면에 기재된 것을 포함할 수 있으나 이로 제한하는 것은 아니다.In addition, nucleic acid molecules encoding a single-chain recombinant FVIII according to the present application can be cloned into various expression vectors for various purposes. The specific configuration of the expression vector may vary depending on the host cell to which the single-chain recombinant FVIII according to the present invention is to be expressed. However, sequences, such as promoters, which regulate the expression of the nucleic acid molecule into mRNA or the protein into protein according to the present application; And / or control sequences such as enhancers. Various vectors and control sequences are known that can be used for these or various other purposes, and those skilled in the art will be able to select appropriate ones in view of the specific purposes and effects herein, including, for example, those described in the Examples and Drawings herein. You can, but are not limited to this.
본원에 따른 재조합 단쇄 FVIII를 코딩하는 핵산분자를 포함하는 벡터는 당업계의 공지된 방법을 통하여 제조될 수 있으며, 예를 들면 본원에 따른 재조합 단쇄 FVIII를 코딩하는 핵산분자를 벡터의 프로모터 및/또는 인핸서에 작동가능하게 연결한다. 일 구현예에서는 세포에서 외래 유전자를 발현시킬 수 있는 재조합 발현 벡터에 삽입되며, 이러한 벡터의 예로는 pMSGneo, pcDNA3.1(+) 등 일반 단백질 발현 벡터를 들 수 있으나, 이에 제한되지 않는다. pMSGneo 벡터의 경우 핵 기질(nuclear matrix)에 결합하는 MAR(Matrix attachment region) 인자가 포함된 발현 벡터로서, 상기 MAR 인자는 발현 벡터에 사용되는 경우 위치-독립적 발현(position-independent expression)을 유도하여 유전자 발현을 증대시키는 역할을 한다. 따라서, pMSGneo 벡터를 이용하는 경우 안정적이고 높은 발현량을 얻을 수 있다. 또한, pcDNA3.1(+) 벡터는 강력한 CMV 프로모터가 함유되어 있어 단백질 발현에 널리 사용되고 있다.A vector comprising a nucleic acid molecule encoding a recombinant single chain FVIII according to the present disclosure may be prepared by methods known in the art, for example, a nucleic acid molecule encoding a recombinant single chain FVIII according to the present disclosure may be a promoter and / or Operate connection to the enhancer. In one embodiment is inserted into a recombinant expression vector capable of expressing a foreign gene in the cell, examples of such vectors include, but are not limited to, general protein expression vectors such as pMSGneo, pcDNA3.1 (+). In the case of the pMSGneo vector, an expression vector including a matrix attachment region (MAR) factor that binds to a nuclear matrix is used. When the MAR factor is used in an expression vector, the MAR factor induces position-independent expression. It plays a role in increasing gene expression. Therefore, when using the pMSGneo vector, a stable and high expression amount can be obtained. In addition, pcDNA3.1 (+) vector contains a strong CMV promoter, widely used for protein expression.
또한, 본원에 따른 재조합 단쇄 FVIII를 코딩하는 핵산분자를 포함하는 상기 재조합 발현 벡터는 다양한 목적을 위해 하기에 기술한 바와 같은 적절한 숙주세포에 형질전환 될 수 있다. 벡터에 포함된 핵산분자를 단백질로 발현하기 위해, 단백질을 코딩하는 핵산분자의 코딩 서열을 목적하는 세포에 적합하게 최적화 할 수 있다.In addition, the recombinant expression vector comprising a nucleic acid molecule encoding a recombinant single-chain FVIII according to the present application can be transformed into a suitable host cell as described below for various purposes. In order to express the nucleic acid molecule contained in the vector as a protein, the coding sequence of the nucleic acid molecule encoding the protein can be optimized appropriately for the desired cell.
이런 측면에서 본원은 본원에 따른 벡터로 전달이입 또는 형질전환된 재조합세포에 관한 것이다. 이러한 세포는 본원에 따른 벡터의 증폭 및/또는 벡터에 포함된 핵산분자가 발현되어, 목적하는 단백질 생산에 사용될 수 있는, 원핵 및 진핵 세포를 모두 포함하는 것이다. 이러한 목적으로 사용될 수 있는 다양한 세포가 공지되어 있으며, 당업자라면 본원의 구체적 목적 및 효과를 고려하여 적절한 것으로 선택할 수 있을 것이며, 예를 들면 본원의 실시예 및 도면에 기재된 것을 포함할 수 있으나 이로 제한하는 것은 아니다. 예를 들면 대장균(E.coli), 포유동물 세포(Mammalian cell), 효모(Yeast), 식물 세포(Plant cell), 곤충 세포(Insect cell)를 포함한다. 본원에 따른 일 구현예에서는 재조합 FVIII의 발현을 위해, 진핵세포, 특히 포유동물 세포주가 사용된다. 당업자라면 본원에 따른 재조합 FVIII의 특징을 고려하여 이러한 특징을 갖는 단백질을 생산할 수 있는 적절한 세포주를 선별할 수 있을 것이며, 예를 들면 이러한 포유동물 세포주로는 당업계에 공지된 CHO, BHK, COS7, HEK 세포주 등을 사용할 수 있으며, 바람직하게는, CHO 세포주, 특히 CHO-S 세포주, CHO-DG44 또는 CHO-K1 세포주, 또는 HEK 세포주 특히 HEK293 세포주가 사용될 수 있으나 이로 제한하는 것은 아니다.In this aspect, the present application relates to recombinant cells transfected or transformed into a vector according to the present application. Such cells include both prokaryotic and eukaryotic cells that can be used for the amplification of the vector and / or nucleic acid molecules contained in the vector, thereby being used to produce the desired protein. Various cells that can be used for this purpose are known, and those skilled in the art will be able to select appropriate ones in view of the specific purposes and effects herein, and may include, for example, those described in the Examples and the drawings herein, but not limited thereto. It is not. Examples include E. coli, Mammalian cells, Yeast, Plant cells, Insect cells. In one embodiment according to the invention eukaryotic cells, in particular mammalian cell lines, are used for expression of recombinant FVIII. Those skilled in the art will be able to select suitable cell lines capable of producing proteins having these characteristics in view of the characteristics of the recombinant FVIII according to the present application. For example, such mammalian cell lines include CHO, BHK, COS7, HEK cell lines and the like can be used, and preferably, CHO cell line, in particular CHO-S cell line, CHO-DG44 or CHO-K1 cell line, or HEK cell line especially HEK293 cell line can be used, but not limited thereto.
본원에 따른 벡터는 발현을 위해 상기와 같은 숙주세포로 전달된다. 이러한 숙주세포로의 벡터의 전달 방법은 당업계에 알려져 있으며, 예를 들면, 칼슘포스페이트 침전법, 샷건 방법, 리포좀을 이용한 방법, 나노니들 또는 전기천공법 등 당업계의 공지된 방법을 이용하여 수행될 수 있으나, 이로 제한하는 것은 아니다.The vector according to the present invention is delivered to such a host cell for expression. Methods for delivering vectors to such host cells are known in the art, for example, calcium phosphate precipitation method, shotgun method, liposome method, using a method known in the art such as nanoneedle or electroporation method May be, but is not limited to such.
본원에 따른 단쇄 FVIII는 반감기의 상승을 목적으로 친수성 폴리머로 특정잔기에서 개질 된 것일 수 있다.Short-chain FVIII according to the present application may be modified at a specific residue with a hydrophilic polymer for the purpose of increasing the half-life.
B 영역의 단백질 구조는 밝혀지지 않았다. B 영역은 구조가 유연하고 당화 정도가 심한 단백질로 음전하가 풍부한 것은 알려져 있으나 그 구조를 예측하기는 어렵다. 즉 분자 모델링 기법을 사용하여 B 영역의 구조를 예측하는 것이 이론적으로 가능하나 B 영역의 후번역 변형 및 당화에 의한 비균질성(heterogeneity)을 고려하면, B 영역 구조를 예측하기 어렵다. 이와 같이 B 영역 내의 개질 위치를 선정하기 위해서는 B 영역 단백질 구조의 표면에 대한 구조 정보가 필요하나 이에 대한 정보가 없어 페길화 위치를 선정하기는 매우 어렵다. The protein structure of the B region is not known. The B region is a protein with a flexible structure and a high degree of glycosylation, and is known to be rich in negative charge, but its structure is difficult to predict. That is, it is theoretically possible to predict the structure of the B region using molecular modeling techniques, but considering the heterogeneity due to post-translational modification and glycosylation of the B region, it is difficult to predict the B region structure. As such, in order to select a modified position in the B region, structural information on the surface of the B region protein structure is required, but since there is no information about this, it is very difficult to select a PEGylation position.
하지만 본원에서는 특히 단쇄 FVIII의 활성에 영향을 주지 않는 A 영역 및/또는 B 영역의 잔기를 예기치 않은 노력을 통하여 선정하고 이를 친수성 폴리머로 개질한 결과 고유의 활성은 유지하면서도 반감기가 향상된 단쇄 FVIII를 수득하였다.However, herein, the residues of region A and / or B which do not particularly affect the activity of short-chain FVIII are selected through unexpected efforts and modified with hydrophilic polymers to obtain short-chain FVIII with improved half-life while maintaining inherent activity. It was.
본원에서는 특히 B 영역의 당화 인근의 잔기를 기준(0)으로 -6 ~ +6 잔기 사이를 페길화 위치로 선정하였다. In the present application, among the residues near the glycosylation of the B region, the PEGylation position was selected between -6 and +6 residues.
이러한 측면에서 본원은 A 영역 및/또는 B 영역 단편의 일부 아미노산 잔기에서 친수성 폴리머와 컨쥬게이션되고, 상기 컨쥬게이션되는 위치는 상기 B 영역에서 서열번호 0의 서열을 기준으로 아미노산 잔기 754, 781, 782, 788, 789, 825 및 897로 구성되는 군으로부터 선택되는 하나 이상의 위치이고; 상기 A-영역에서 서열번호 0의 서열을 기준으로 아미노산 잔기 491, 495, 498 및 1806으로 구성되는 군으로부터 선택되는 하나 이상의 위치이고, 상기 컨쥬게이션 되는 위치의 잔기는 상기 친수성 폴리머와의 접합을 위해 시스테인으로 치환되는, 단쇄 혈액응고 8인자에 관한 것이다. In this aspect, the present application is conjugated with a hydrophilic polymer at some amino acid residues of the A region and / or B region fragment, and the conjugated position is based on the amino acid residues 754, 781, 782 in the B region based on the sequence of SEQ ID NO: 0. At least one position selected from the group consisting of 788, 789, 825 and 897; At least one position selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 0 in the A-region, wherein the residue at the conjugated position is for conjugation with the hydrophilic polymer A single-chain coagulation factor 8 is substituted with cysteine.
본원에 따른 단쇄 FVIII의 개질에 사용될 수 있는 친수성 폴리머는 당업계의 공지된 다양한 폴리머가 사용될 수 있으며, 예를 들면 폴리에틸렌글리콜(Polyethylene glycol), 폴리에틸렌 옥사이드, 덱스트란, 폴리시알릭 등을 포함하나 이로 제한하는 것은 아니다. 이들 폴리머는 생체 적합성(비독성, 저독성) 친수성의 유연한 구조이고 단백질에 화학적으로 접합 시 접합 된 단백질이 비특이적인 상호 작용(interaction)으로 타 분자 및 물체의 표면에 붙어 활성이 소실되는 것을 방지하고 혈 중에서 단백질 분해 효소에 의해 비 활성화되는 것을 방지하는 역할을 한다. Hydrophilic polymers that can be used for the modification of short-chain FVIII according to the present application can be used a variety of polymers known in the art, including, but not limited to, polyethylene glycol, polyethylene oxide, dextran, polysialic, etc. It is not. These polymers are biocompatible (non-toxic, low-toxic) hydrophilic, flexible structures that prevent the loss of activity and prevent loss of activity due to non-specific interactions of the conjugated proteins with chemically conjugated proteins. It serves to prevent inactivation by proteolytic enzymes.
본원에 따른 일 구현 예에서는 PEG가 사용되며, 특히 20kDa 이상의 PEG가 사용된다. PEG는 단백질에 접합되어 단백질 주위를 감싸는 효과를 부여함으로써 체내에서 scavenger receptor에 결합 후 소실되거나 inactivating protease에 의한 분해에 방어할 수 있게 된다. PEG는 사슬 모양의 고분자로 선형(linear) 또는 가지형(branched)으로 되어 있으며 작은 분자량의 PEG는 단백질을 충분히 감싸지 못하여 단백질을 완전히 방어하지 못한다. 그러므로 방어하고자 하는 단백질을 충분히 감싸기 위해서는 한계 수준이상의 분자량이어야 한다. 본원의 단쇄 FVIII의 페길화는 페길화 위치의 아미노산을 시스테인으로 치환 후 시스테인에 특이적인 acryloyl, sulfone 또는 maleimide기를 한쪽 끝에 포함하는 PEG를 통해 접합 될 수 있다. In one embodiment according to the invention PEG is used, in particular PEG of at least 20 kDa. PEG binds to proteins and gives them the effect of wrapping around the protein, which can then be lost after binding to scavenger receptors in the body or to prevent degradation by inactivating protease. PEG is a chain-shaped polymer that is linear or branched, and small molecular weight PEG does not cover the protein enough to completely protect the protein. Therefore, the molecular weight must be above the limit level to sufficiently cover the protein to be protected. PEGylation of the short-chain FVIII of the present application may be conjugated via PEG containing one end of an acryloyl, sulfone or maleimide group specific to cysteine after substitution of an amino acid at the PEGylation position with cysteine.
즉, 본원에 따른 단쇄 FVIII은 친수성 폴리머로의 개질을 위해 특정 잔기가 치환될 수 있으며, 구체적으로 치환되는 잔기는 단쇄 FVIII 상에서 친수성 폴리머가 접합되는 위치에 따라 상이하며, 당업자라면 친수성 폴리머의 접합을 위하여 적절한 잔기로 개질 할 수 있을 것이다. That is, the short-chain FVIII according to the present application may be substituted with a specific residue for modification to the hydrophilic polymer, specifically, the residue to be replaced depends on the position where the hydrophilic polymer is conjugated on the short-chain FVIII, and those skilled in the art May be modified with appropriate residues.
본원에 따른 일 구현예에서는 개질로서 페길화가 사용되며, 이 경우 개질되는 잔기는 시스테인으로 치환된다. In one embodiment according to the invention PEGylation is used as the modification, in which case the residue to be modified is substituted with cysteine.
본원에 따른 일 구현예에서는 A2 영역 495번 발린 잔기 및/또는 B 영역의 782번 아이소루이신 잔기에서 개질되며, 특히 페길화되고, 페길화를 위해 상기 잔기가 시스테인으로 치환된다. In one embodiment according to the invention is modified at the A2 region 495 valine residue and / or B region 782 isoleucine residue, in particular PEGylated, the residue is substituted with cysteine for PEGylation.
개질을 위해 시스테인으로 치환된 단백질의 발현은 당업계에 공지된 방법을 이용하여 수행될 수 있으며, 예를 들면 페길화되는 단백질의 경우, 도입된 자유 시스테인은 세포 내의 발현 도중이나 세포 밖으로 배출 후 free thiol을 포함하는 저분자 물질인 시스테인 또는 글루타치온과 이황화 결합을 형성하여 도입된 자유 시스테인을 마스킹하여 페길화 직전 환원 조건으로 도입된 자유 시스틴이 복원되기 전까지 도입된 자유시스틴을 안정화시켜 페길화 효율을 높이는 것이 필요하다.Expression of a protein substituted with cysteine for modification can be performed using methods known in the art, for example, for proteins to be PEGylated, free cysteine introduced is free during expression in cells or after exiting cells. Masking free cysteine introduced by forming disulfide bonds with cysteine or glutathione, a low-molecular substance containing thiol, and stabilizing the free cystine introduced by reducing conditions immediately before PEGylation to increase PEGylation efficiency need.
페길화 반응은 공지된 방법을 이용하여 수행될 수 있으며, 예를 들면 자유 시스테인의 마스킹을 위해 사용된 시스테인이나 글루타치온은 페길화 반응 전에 제거되어 도입한 시스틴의 free thiol이 복원되어야 하며, 이를 위해 TCEP, DTT, beta-mercaptoethanol, 시스테인(cysteine), 글루타티온(환원형) 등의 환원제를 처리하는 과정과 이 과정에서 FVIII 본연의 이황화본드가 환원적으로 절단된 것을 복원하기 위한 산화 과정을 포함하여 수행할 수 있다.The PEGylation reaction can be carried out using a known method, for example, cysteine or glutathione used for masking free cysteine must be removed before the PEGylation reaction to restore the free thiol of cystine introduced. , Including the treatment of reducing agents such as DTT, beta-mercaptoethanol, cysteine, glutathione (reduced form), and oxidation to restore the reductive cleavage of the native disulfide bonds in this process. Can be.
발현된 재조합 단백질의 도입된 시스테인(cysteine)의 매스킹은 공지된 방법을 이용하여 수행될 수 있으며, 예를 들어 자유 시스테인의 마스킹을 위해 사용된 시스테인이나 글루타치온은 세포 배양 배지 성분에 추가로 첨가하거나, 세포 배양 배지 성분에 추가적인 첨가 없이 세포의 성장과 유지 과정에서 정상적으로 세포가 생성하여 축적하고 분비하는 시스테인 이나 글루타치온 성분이 사용될 수 있다. Masking of the introduced cysteine of the expressed recombinant protein can be performed using known methods, for example cysteine or glutathione used for masking free cysteine in addition to the cell culture medium components or In addition, cysteine or glutathione components, which are normally produced, accumulated and secreted by cells during growth and maintenance of cells, may be used without additional addition to cell culture medium components.
다른 측면에서 본원은 또한 상술한 본원에 따른 벡터를 진핵세포에 전달이입하는 단계; 상기 세포를 배양액 중에서 배양하는 단계; 상기 배양액을 수집하여 시스테인(cysteine) 도입 FVIII을 정제하는 단계; 및 상기 정제된 시스테인(cysteine) 도입 FVIII을 페길화 완충액으로 처리하는 단계; 도입된 시스테인(cysteine)의 자유 티올기를 환원 과정을 통해 복원하는 단계; 환원 과정 중 분리된 단쇄 FVIII 단백질 내부의 이황화 결합을 복원하기 위하여 산화시키는 단계; 환원형의 티올기로 복원된 단쇄 FVIII 시스테인(cysteine)에 특이적으로 페길화시키는 단계 및 페길화 단쇄 FVIII을 분리하는 단계를 포함하는, 단쇄 응고인자 8의 생산방법에 관한 것이다. In another aspect, the present invention also provides a method of transfecting a vector according to the present application into an eukaryotic cell; Culturing the cells in culture; Collecting the culture solution to purify cysteine-introduced FVIII; And treating the purified cysteine introduced FVIII with PEGylation buffer; Restoring the introduced free thiol group of cysteine through a reduction process; Oxidizing to restore disulfide bonds within the isolated short chain FVIII protein during the reduction process; The present invention relates to a method for producing short-chain coagulation factor 8, comprising specifically PEGylating a short-chain FVIII cysteine restored to a reduced thiol group and isolating PEGylated short-chain FVIII.
본원에 따른 방법에 사용되는 구체적인 벡터, 세포 및 각 단계의 처리 과정은 본원 실시예에 기재된 것을 참고 할 수 있다. Specific vectors, cells, and treatment steps for each step used in the method according to the present application can be referred to those described in the Examples herein.
본원에 따른 재조합 단쇄 FVIII는 혈액응고가 필요한 질환, 혈우병, 특히 혈우병 A 환자의 혈액응고 또는 혈우병 A의 치료에 유용하게 사용될 수 있다. Recombinant single-chain FVIII according to the present invention can be usefully used for the treatment of hemophilia A, hemophilia, in particular hemophilia A patients, or hemophilia A.
이에 본원은 또한, 본원의 청구범위를 포함하는 본원에 개시된 어느 하나의 단쇄 혈액응고 8인자를 포함하는 혈우병 환자 또는 혈액의 응고가 필요한 환자에 사용하기 위한 혈액 응고 키트, 상기 단쇄 혈액응고 8인자의 혈우병 치료용 용도 또는 조성물, 또는 단쇄 혈액응고 8인자를 암호화하는 염기 서열의 핵산을 포함하는 조성물 사용하는 혈우병 A형의 유전 치료 용도 및 그 치료에 사용될 단쇄 혈액응고 8인자를 암호화하는 염기 서열의 핵산을 포함하는 조성물 또는 단쇄 혈액응고 8인자를 혈우병 환자 또는 혈액의 응고가 필요한 환자에게 치료적으로 유효한 양으로 투여하는 단계를 포함하는 혈우병 치료 방법 또는 혈액응고 방법에 관한 것이다.Accordingly, the present application also provides a blood coagulation kit for use in a hemophilia patient or a patient in need of blood coagulation, including any one of the short chain coagulation factors 8 disclosed herein, including the claims of the present application, Use or composition for the treatment of hemophilia, or a composition comprising a nucleic acid of a base sequence encoding a single-chain coagulation factor 8 Nucleic acid of the hemophilia type A using the composition and the nucleic acid of the base sequence encoding a single-chain coagulation factor 8 to be used in the treatment It relates to a hemophilia treatment method or a blood coagulation method comprising the step of administering to the hemophilia patients or patients in need of coagulation of the blood containing a composition or a short-chain coagulation factor eight.
본원에 따른 재조합 단쇄 FVIII는 약학적으로 허용가능한 담체를 함께 포함하는 혈우병 치료용 약학 조성물의 형태로 제공될 수 있다. Recombinant single-chain FVIII according to the present application may be provided in the form of a pharmaceutical composition for treating hemophilia comprising a pharmaceutically acceptable carrier.
또한 본원의 약학 조성물은 단독으로, 또는 기타 약물치료 및 생물학적반응조절제를 사용하는 방법들과 병용하여 사용할 수 있다. In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using other drug treatments and biological response modifiers.
본원의 조성물은 상기 언급한 유효성분 이외에 추가로 약학적 또는 생리학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. The composition of the present invention may be prepared by including one or more pharmaceutically or physiologically acceptable carriers in addition to the above-mentioned active ingredients.
본원에서 사용된 용어 담체란 사용되는 투여량 및 농도에 노출되는 세포 또는 포유동물에 무독성인 약학적으로 허용가능한 담체, 부형제, 또는 안정화제를 의미하는 것이다. 이러한 담체의 예로는 식염수, 링거액, 완충 식염수, 포스페이트, 시트레이트 및 다른 유기산과 같은 완충액, 아스코르브산을 비롯한 산화방지제, 저분자량(약 10 잔기 미만) 폴리펩타이드, 단백질, 예를 들어 혈청 알부민, 젤라틴 또는 면역글로불린; 친수성 중합체, 예를 들어폴리비닐피롤리돈, 아미노산, 예를 들어 글리신, 글루타민, 아스파라긴, 아르기닌 또는 라이신, 단당류, 이당류 및 글루코스, 만노스 또는 덱스트린을 비롯한 기타 탄수화물, 킬레이트화제, 예를 들어 EDTA, 당 알콜, 예를 들어 만니톨 또는 소르비톨, 염 형성 카운터 이온, 예를 들어 나트륨, 및(또는) 비이온계 계면활성제, 예를 들어 트윈, 폴리에틸렌 글리콜(PEG) 및 플루로닉스(PLURONICS)를 들 수 있다. The term carrier, as used herein, means a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to a cell or mammal that is exposed to the dosages and concentrations employed. Examples of such carriers include saline, Ringer's solution, buffered saline, buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin Or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, for example EDTA, sugars Alcohols such as mannitol or sorbitol, salt-forming counter ions such as sodium, and / or nonionic surfactants such as tween, polyethylene glycol (PEG) and PLURONICS.
필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형으로 제제화할 수 있다. 더 나아가 당해 기술분야의 적정한 방법으로 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제형화할 수 있다. If desired, other conventional additives such as antioxidants, buffers, bacteriostatics, and the like may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.
본원의 조성물은 목적하는 방법에 따라 특히 비경구투여(예를 들어, 정맥 내, 피하, 복강 내)가 바람직하다. 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The compositions herein are particularly preferred parenteral administration (eg, intravenous, subcutaneous, intraperitoneal) according to the desired method. The dosage depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the time of day, and may be appropriately selected by those skilled in the art.
본원에 따른 조성물은 치료적으로 유효한 양으로 투여된다. 본원에서, "치료적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the invention is administered in a therapeutically effective amount. As used herein, "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with an effective dose level being the type of disease, severity, activity of the drug, drug Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
본원에 따른 재조합 단쇄 FVIII는 그 자체 또는 상술한 바와 같은 조성물의 형태로 혈우병 또는 혈액응고가 필요한 대상체에게 치료적으로 유효한 양으로 투여되어, 혈액 응고 방법 또는 혈우병의 치료 방법의 형태로 제공될 수 있으며, 앞서 기재한 바를 참고할 수 있다. Recombinant single-chain FVIII according to the present application may be administered in a therapeutically effective amount to a subject in need of hemophilia or coagulation in the form of a composition as described above or in the form of a blood coagulation method or a method of treating hemophilia, This may be referred to the above description.
본원에서 사용되는 용어, "치료"란 본 발명의 조성물의 투여에 의해 질환에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. 본원에 따른 방법이 사용되는 대상체는 인간을 포함하는 영장류이나, 이로 제한하는 것은 아니다.As used herein, the term "treatment" means any action in which symptoms caused by a disease are improved or beneficially altered by administration of a composition of the present invention. Subjects in which the methods according to the invention are used are primates including but not limited to humans.
이하, 본 발명의 이해를 돕기 위해서 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 하기의 실시예에 한정되는 것은 아니다. Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.
실시예 Example
실시예 1. 단쇄(Single-chain) FVIII 구축Example 1 Single-chain FVIII Construction
인간 혈액 응고 인자 VIII(Factor VIII) 고유의 특성을 유지하면서도 발현율이 높은 단쇄 형태의 인간 혈액 응고 인자 FVIII(single-chain FVIII, scFVIII)을 개발하기 위하여, FVIII의 a3를 포함하는 B 영역 길이를 조절하여 다양한 길이의 B 영역을 포함하는 재조합 scFVIII을 제작하여 발현하고 그 발현량과 활성(APTT, CS)을 분석하였다. 상세한 방법 및 결과는 아래와 같다. In order to develop a single-chain high-expression human blood coagulation factor FVIII (scFVIII) while maintaining the characteristics of human blood coagulation factor VIII, the length of the B region including a3 of FVIII is controlled. The recombinant scFVIII including the B region of various lengths was prepared and expressed, and its expression and activity (APTT, CS) were analyzed. Detailed methods and results are as follows.
실시예 1-1. scFVIII 제작Example 1-1. scFVIII production
단쇄 형태로 발현되는 FVIII의 구축을 위해, furin 절단 위치인 1648 잔기(전장 FVIII 아미노산 서열을 나타내는 서열번호 0의 아미노산 서열기준)를 포함하는 B 영역의 일부가 결실된 FVIII를 구축하였다. 중쇄와 경쇄를 연결하는 B 영역 길이에 따라 최종 FVIII의 활성과 특성에 영향을 줄 수 있기 때문에, scFVIII에 포함되는 B 영역 길이 결정을 위해 표 1에 기재된 바와 같이 총 7종류의 FVIII를 구축하였다. 구축된 각각의 scFVIII은 B 영역의 N-terminal(서열번호 1의 741번째 아미노산 잔기)로부터 특정 길이의 B 영역을 포함하고 이러한 B 영역이 원래 보유하고 있는 당쇄를 그대로 포함하도록 제작되었다.For the construction of FVIII expressed in the short chain form, FVIII was constructed in which a part of the B region including the furin cleavage position 1648 residue (based on the amino acid sequence of SEQ ID NO: 0 representing the full length FVIII amino acid sequence) was deleted. Since the length and length of the B region connecting the heavy and light chains may affect the activity and properties of the final FVIII, a total of seven types of FVIII were constructed as shown in Table 1 to determine the length of the B region included in the scFVIII. Each scFVIII constructed was constructed to include a B region of a certain length from the N-terminal (SEQ ID NO: 741 amino acid residue) of the B region and to contain the sugar chain originally possessed by this B region.
또한 경쇄와 중쇄 부위를 연결하는 B 영역에 의한 구조적인 방해를 최소화하여 천연형의 FVIII 활성과 유사한 특성을 갖도록 하기 위해, 경쇄 부위와 인접한 a3 domain의 일부가 다른 구조의 scFVIII 보다 많이 포함된 구조를 또한 제작하였다 (서열번호 6). 또한 CHO 세포에서 발현을 향상시키기 위하여 CHO 코돈으로 최적화된 scFVIII를 제작하였다 (핵산 서열번호 15). In addition, in order to minimize structural interference caused by the B region connecting the light and heavy chain sites to have properties similar to those of the native FVIII activity, a portion of the a3 domain adjacent to the light chain site is contained more than scFVIII of other structures. Also prepared (SEQ ID NO: 6). In addition, scFVIII optimized with CHO codon was made to enhance expression in CHO cells (nucleic acid SEQ ID NO: 15).
B 영역이 결실된 각 scFVIII은 인간 FVIII 핵산서열(NM000132)을 기준으로 합성하였다. 먼저 scFVIII 유전자 발현벡터를 제조하기 위하여 인간 FVIII 핵산서열(NM000132)을 기준으로 G1 단백질 서열(Leader-Heavy chain-B domin (741-764, 1653-1689)-Light chain)을 암호화(인코딩)하고 5’에 NotI-AsisI과 3’에 XhoI-PacI 효소절단 부위를 포함하도록 GeneArt 사를 통해 유전자 전체를 합성하였다. 합성된 유전자를 pcDSW 벡터의 NotI/XhoI site에 클로닝하여 pcDSW-G1 발현 벡터를 구축하였고, 발현 벡터 구축의 정확성은 AsisI/PacI 효소를 이용하여 절단 후 절단된 밴드 크기 비교를 통해 확인하였다. 이후 B 영역이 결실된 각 scFVIII (G2, G3, G4, G4-flex, G6)는 FVIII heavy chain의 A2 영역 내에 존재하는 BamHI site (GGATCC)부터 light chain 끝에 PacI 효소절단 부위를 포함하도록 GeneArt 사를 통해 각각의 scFVIII들에 해당하는 유전자를 합성한 후 기존에 완성된 pcDSW-G1 발현벡터를 효소 BamHI/PacI을 이용하여 기존 FVIII의 해당 부위를 제거한 후 합성된 유전자를 BamHI/PacI site로 자르고 pcDSW-G1 발현벡터의 BamHI/PacI site에 클로닝 하여 pcDSW-scFVIII 발현 벡터를 구축하였고, 발현 벡터 구축의 정확성은 BamHI/PacI 효소를 이용하여 절단 후 크기 비교를 통해 확인하였다. 발현을 향상시키기 위하여 CHO 코돈으로 최적화된 scFVIII(G6_opt)은 GeneArt 사를 통해 5’에 NotI-AsisI과 3’에 XhoI-PacI 효소절단 부위가 포함되고 동물세포 발현에 적합하도록 코돈 최적화 및 유전자 합성을 완료하였다. 합성된 유전자를 pcDSW 벡터의 NotI/XhoI site에 클로닝 하여 발현 벡터를 구축하였고, 발현 벡터 구축의 정확성은 BamHI 효소를 이용하여 절단 후 크기 비교를 통해 확인하였다.Each scFVIII lacking the B region was synthesized based on the human FVIII nucleic acid sequence (NM000132). First, the G1 protein sequence (Leader-Heavy chain-B domin (741-764, 1653-1689) -Light chain) was encoded based on the human FVIII nucleic acid sequence (NM000132) to prepare the scFVIII gene expression vector. Genes were synthesized through GeneArt to include Xho I- Pac I enzyme cleavage sites in Not I- Asis I and 3 '. The synthesized gene was cloned into the Not I / Xho I site of the pcDSW vector to construct a pcDSW-G1 expression vector, and the accuracy of the expression vector construction was confirmed by comparing the cut band sizes after cleavage using the Asis I / Pac I enzyme. It was. Each scFVIII (G2, G3, G4, G4-flex, G6) is deleted from the BamH I site (GGATCC) in the A2 region of the FVIII heavy chain from the gene chain to include the Pac I enzyme cleavage site at the end of the light chain. After synthesizing the genes corresponding to the respective scFVIII through the use of the previously completed pcDSW-G1 expression vector using the enzyme BamH I / Pac I to remove the corresponding region of the existing FVIII and synthesized gene BamH I / Pac A pcDSW-scFVIII expression vector was constructed by cutting to the I site and cloning to the BamH I / Pac I site of the pcDSW-G1 expression vector, and the accuracy of the expression vector construction was confirmed by comparing the sizes after cleavage using the BamH I / Pac I enzyme. It was. ScFVIII (G6_opt) optimized with CHO codons to enhance expression is optimized for codon expression for animal cell expression by GeneArt Inc. including Not I- Asis I at 5 'and Xho I- Pac I enzyme cleavage site at 3' And gene synthesis was completed. The synthesized gene was cloned into the Not I / Xho I site of the pcDSW vector to construct an expression vector, and the accuracy of expression vector construction was confirmed by comparing the sizes after cleavage using a BamH I enzyme.
[표 1] 본원에서 제작된 다양한 길이의 B 영역을 포함하는 scFVIIITABLE 1 scFVIII comprising B regions of various lengths made herein
Figure PCTKR2017006633-appb-I000001
Figure PCTKR2017006633-appb-I000001
* 서열번호 1의 서열을 기준으로 기재* Based on the sequence of SEQ ID NO: 1
** 코딩핵산 서열을 CHO 세포의 코돈으로 최적화 ** Optimizing Coding Nucleic Acid Sequences into Codons of CHO Cells
실시예 1-2. 구축된 scFVIII 발현 및 활성 측정Example 1-2. Constructed scFVIII Expression and Activity Measurement
scFVIII 발현scFVIII expression
실시예 1-1에서 구축된 scFVIII를 세포에서 발현하였으며, 발현된 세포는 세포배양배지로 배양된다. 우선 발현을 위해 실시예 1-1에서 구축된 발현 벡터를 일시발현시스템인 Expi293F™ Expression System Kit (Thermofisher 사, Catalog Number A14635)를 사용하여 Expi293F™ 세포에 전달 이입하였다. 간략히 설명하면 형질전환 수행 24시간 전에 Expi293F™ 세포를 2.0 X 106 cells/mL로 Expi293 배양배지를 이용하여 예상 필요량에 맞추어 계대배양한 후 형질전환 수행 당일, 세포 수와 세포 생존율(viability)을 측정하여 세포 생존율이 95% 이상일 경우 형질전환을 진행하였다. 125mL flask에 7.5 X 107 cell을 Expi293 배양배지를 첨가하여 최종 25.5mL 볼륨으로 맞춰주었다 (30mL 기준). 실시예 1-1에서 구축된 발현 벡터 30μg을 Opti-MEM을 이용하여 총 볼륨이 1500μL이 되도록 섞어주었다. 80μL transfection reagent를 Opti-MEM을 이용하여 총 볼륨이 1500μL이 되도록 섞어주고 상온에서 5분간 incubation 해주었다. 5분 후 transfection reagent가 들어있는 Opti-MEM을 DNA가 들어있는 Opti-MEM에 넣어주고 부드럽게 섞어주었다. 그리고 20~30분간 상온에서 반응시켜 주었다. 3mL의 DNA : transfection reagent 복합체를 미리 준비해 놓은 125mL flask Expi293F™ 세포에 (Total volume : 28.5mL)에 한 방울씩 떨어뜨려 주고 37℃, 5% CO2 shacking incubator에서 125 rpm으로 배양하였다. 16~20시간 후에 Enhancer 1과 Enhancer 2를 각각 150μL, 1.5mL을 넣어주고 34℃, 5% CO2 shacking incubator에서 125 rpm으로 배양하였다. 배양 3일째 배지를 harvest하고 아래와 같이 scFVIII의 활성을 측정하였다.ScFVIII constructed in Example 1-1 was expressed in cells, and the expressed cells were cultured in cell culture medium. First, for expression, the expression vector constructed in Example 1-1 was transfected into Expi293F ™ cells using the transient expression system Expi293F ™ Expression System Kit (Thermofisher, Catalog Number A14635). Briefly, Expi293F ™ cells were passaged at 2.0 X 10 6 cells / mL at an expected requirement 24 hours before transformation, and then the number of cells and viability were measured on the day of transformation. When cell viability was 95% or more, transformation was performed. 7.5 X 10 7 cells were added to a 125 mL flask to adjust the final volume of 25.5 mL by adding Expi293 culture medium (based on 30 mL). 30 μg of the expression vector constructed in Example 1-1 was mixed using Opti-MEM so that the total volume was 1500 μL. 80μL transfection reagent was mixed to a total volume of 1500μL using Opti-MEM and incubated at room temperature for 5 minutes. After 5 minutes, the Opti-MEM containing the transfection reagent was added to the Opti-MEM containing the DNA and mixed gently. And reacted at room temperature for 20-30 minutes. A 3 mL DNA: transfection reagent complex was previously dropped in a 125 mL flask Expi293F ™ cell (Total volume: 28.5 mL) and incubated at 37 rpm, 125 rpm in a 5% CO 2 shacking incubator. After 16 to 20 hours, 150 μL and 1.5 mL of Enhancer 1 and Enhancer 2 were added thereto, and then incubated at 34 rpm and 125 rpm in a 5% CO 2 shacking incubator. On day 3 of culture, the medium was harvested and scFVIII activity was measured as follows.
발현된 scFVIII 활성 측정Determining expressed scFVIII activity
현재 전장 형태와 B 영역 결실 형태 2종류의 재조합 FVIII이 임상적으로 사용되고 있다. 하지만 이들 두 종류 재조합 FVIII이 임상적으로 활성 및 효력에서 동등한지 이슈가 제기되고 있다 (GRUPPO, R. A. et al.. 2003. Comparative effectiveness of full-length and B-domain deleted factor VIII for prophylaxis-a metaanalysis. Haemophilia, 9, 251-60; LOLLAR, P. 2003. The factor VIII assay problem: neither rhyme nor reason. J Thromb Haemost, 1, 2275-9; MIKAELSSON, M. et al., 2001. Measurement of factor VIII activity of B-domain deleted recombinant factor VIII. Semin Hematol, 38, 13-23). Currently, two types of recombinant FVIII, full length form and B region deletion form, are used clinically. However, an issue has been raised whether these two recombinant FVIII are clinically equivalent in activity and potency (GRUPPO, RA et al .. 2003. Comparative Effectiveness of full-length and B-domain deleted factor VIII for prophylaxis-a metaanalysis. Haemophilia, 9, 251-60; LOLLAR, P. 2003.The factor VIII assay problem: neither rhyme nor reason.J Thromb Haemost, 1, 2275-9; MIKAELSSON, M. et al., 2001.Measurement of factor VIII activity of B-domain deleted recombinant factor VIII.Semin Hematol, 38, 13-23).
BDD-FVIII은 응고분석(APTT)으로 측정한 활성이 chromogenic assay(CS)로 측정한 활성보다 50%까지 낮은 반면 전장 형태의 FVIII은 두 가지 측정값이 유사한 것으로 나타났다 (BARROWCLIFFE et al., SEMIN THROMB HEMOST, vol. 28(3), 2002: 47 - 56). Clotting assay에서 BDD의 낮은 활성은 실제 long-term prophylaxis에서 BDD-FVIII이 전장 FVIII 보다 A형 혈우병 환자의 출혈을 예방하지 못하는 이유로 제기되고 있다 (GRUPPO, R. A., et al., 2003. Comparative effectiveness of full-length and B-domain deleted factor VIII for prophylaxis--a metaanalysis. Haemophilia, 9, 251-60; GRUPPO, R. A. et al., 2004. Meta-analytic evidence of increased breakthrough bleeding during prophylaxis with B-domain deleted factor VIII. Haemophilia, 10, 747-50; GRUPPO, R. A. et al., 2004. Increased breakthrough bleeding during prophylaxis with B-domain deleted factor VIII-a robust metaanalytic finding. Haemophilia, 10, 449-51).BDD-FVIII was up to 50% lower in activity as measured by coagulation assay (APTT) than in chromogenic assay (CS), while full-length FVIII was similar in both measurements (BARROWCLIFFE et al., SEMIN THROMB HEMOST, vol. 28 (3), 2002: 47-56). The low activity of BDD in clotting assays has been attributed to the fact that BDD-FVIII does not prevent bleeding in patients with hemophilia A than full-length FVIII in real-term prophylaxis (GRUPPO, RA, et al., 2003. Comparative Effectiveness of full -length and B-domain deleted factor VIII for prophylaxis--a metaanalysis.Haemophilia, 9, 251-60; GRUPPO, RA et al., 2004. Meta-analytic evidence of increased breakthrough bleeding during prophylaxis with B-domain deleted factor VIII Haemophilia, 10, 747-50; GRUPPO, RA et al., 2004. Increased breakthrough bleeding during prophylaxis with B-domain deleted factor VIII-a robust metaanalytic finding.Haemophilia, 10, 449-51).
따라서 본원에서는 전장 재조합 FVIII와 유사한 수치의 비(specific)활성(CS, APTT)과 비활성 비율(APTT/CS)을 갖는 scFVIII을 선정하기 위하여 실시예 1-1에서 구축된 각각의 scFVIII에 대해 CS와 clotting assay(APTT)로 측정한 비활성 수치와 비활성 비율((APTT)/CS)을 분석하였으며, 이를 scFVIII을 선정하는 기준으로 사용하였다. 비교군으로는 실제 생체 내의 FVIII와 유사한 전장, 이쇄 형태의 재조합 FVIII(Advate®, Baxalta) 제품을 비교 기준으로 사용하였다.Therefore, in the present application, for each scFVIII constructed in Example 1-1 to select scFVIII having a specific activity (CS, APTT) and inactivity ratio (APTT / CS) similar to the full length recombinant FVIII, CS and Inactivation levels and inactivation ratios ((APTT) / CS) measured by clotting assay (APTT) were analyzed and used as the criteria for selecting scFVIII. In the comparison group, a full-length, double-chain recombinant FVIII (Advate®, Baxalta) product similar to FVIII in vivo was used as a reference.
구체적으로 본원에서 사용한 FVIII 활성 측정 시험 방법은 one-stage method와 chromogenic method 두 가지이다. One-stage method는 FVIII deficient plasma, activator, phospholipids와 FVIII 시료를 섞어 준 후, 혈액응고 시간을 측정하여 시료의 FVIII 활성을 측정하는 방법이다. Chromogenic method는 FIXa, FX, thrombin, calcium, phospholipids와 FVIII 시료를 섞어 준 후, FVIII 시료에 의해 활성화된 FXa의 양을 측정하기 위해 FXa에 의해 잘리면서 발색하는 chromogenic substrate를 넣어주고, chromogenic substrate의 발색 정도에 따라 FVIII의 활성을 측정하는 방법이다. 이 두 방법의 차이는 one-stage method는 FVIII deficient plasma를 이용하고, 응고(clotting) 시간을 측정하는 것인데, chromogenic method는 FVIII deficient plasma를 이용하는 것이 아닌, FVIII의 activation에 필요한 인자와 활성화된 FVIII을 측정하기 위한 기질만 넣어준 후, 기질의 발색 정도를 측정하는 것이다.Specifically, the FVIII activity measurement test methods used herein are two-stage method and chromogenic method. One-stage method is a method for measuring FVIII activity of a sample by mixing FVIII deficient plasma, activator, phospholipids and FVIII sample and measuring blood coagulation time. The chromogenic method mixes FIXa, FX, thrombin, calcium, phospholipids and FVIII samples, inserts a chromogenic substrate that is cut and developed by FXa to measure the amount of FXa activated by the FVIII sample, and develops the chromogenic substrate. It is a method of measuring the activity of FVIII according to the degree. The difference between these two methods is that the one-stage method uses FVIII deficient plasma and measures clotting time. The chromogenic method does not use FVIII deficient plasma, but the factors necessary for activation of FVIII and activated FVIII. After adding the substrate for measurement, the degree of color development of the substrate is measured.
One-stage method에 의한 활성 측정은 ACL TOP CTS 500 장비에 미리 만든 자동화된 분석법을 사용하여 다음과 같이 수행하였다. 시료를 FVIII deficient plasma를 사용하여 1 IU/mL 농도로 희석하고, 표준품은 NIBSC FVIII standard vial에 1mL 증류수를 넣어 녹인 후, FVIII deficient plasma를 사용하여 1 IU/mL 농도로 희석하여 준비하였다. 실험 장비의 rack type에 맞추어 소모 시약 및 측정 시료를 준비하였다. ACL TOP CTS 500 장비의 R rack에 순차적으로 calcium chloride vial을 바코드가 인식되도록 놓고, APTT-SP vial에는 magnetic stirring bar를 넣은 후, vial의 바코드가 인식되도록 R rack에 놓았다. FVIII deficient plasma는 측정 시료 개수 × 400μL를 준비하여 vial이 인식되지 않도록 주변부를 테이프를 붙이고 R rack에 놓았다. DA rack에는 G.O. buffer (Imidazole 3.4g, NaCl 5.8g, 1L, pH 7.4) 50mL에 알부민 2.63mL을 첨가한 vial을 놓았다. Sample cup에 앞에서 준비한 시료와 표준품을 400μL 이상 넣은 후, sample rack에 sample cup을 놓는다. ACL TOP program에서 미 인식된 시약 및 측정 시료의 정보를 입력하고, 미리 만든 자동화된 분석법을 실행하였다. 자동화 분석법의 모든 반응은 37도에서 다음과 같이 진행되었다. 우선 측정할 시료를 G.O 버퍼로 10배 pre-dilution 후 각각을 1, 3, 10배 더 희석하여 100%, 33,33%, 10% 시료를 만든다. 희석한 시료 50μL를 30-45초간 정치한 후 50μL FVIII deficient plasma와 섞어 준 후 60-70초간 정치시켰다. 다음 50μL (intermediate reagent) APTT-SP를 첨가 후 300-340초 정치하고 마지막으로 50μL start reagent (APTT-SP CaCl2)를 첨가한 후 응고 시간을 측정하였다.Activity measurement by the one-stage method was performed using an automated method previously prepared on the ACL TOP CTS 500 instrument as follows. Samples were diluted to 1 IU / mL concentration using FVIII deficient plasma, and 1 mL distilled water was dissolved in NIBSC FVIII standard vial, and then diluted to 1 IU / mL concentration using FVIII deficient plasma. Consumable reagents and measurement samples were prepared according to the rack type of the experimental equipment. The calcium chloride vial was placed in the R rack of the ACL TOP CTS 500 equipment in order to recognize the barcode, and the magnetic stirring bar was placed in the APTT-SP vial and placed in the R rack so that the barcode of the vial was recognized. FVIII deficient plasma was prepared by measuring the number of samples × 400μL and taped the periphery to Ral to prevent vial recognition. DA rack has G.O. A vial containing 2.63 mL of albumin was placed in 50 mL of buffer (Imidazole 3.4 g, NaCl 5.8 g, 1 L, pH 7.4). Put the sample prepared above and 400μL into the sample cup and place the sample cup in the sample rack. Information from unrecognized reagents and measurement samples was entered in the ACL TOP program, and pre-made automated assays were run. All reactions of the automated assay proceeded as follows at 37 degrees. First, the sample to be measured is pre-dilution 10 times with G.O buffer, and then diluted 1, 3, and 10 times further to make 100%, 33,33%, and 10% samples. 50 μL of the diluted sample was allowed to stand for 30-45 seconds, and then mixed with 50μL FVIII deficient plasma and allowed to stand for 60-70 seconds. After adding 50 μL (intermediate reagent) APTT-SP, the mixture was allowed to stand for 300-340 seconds, and finally, 50 μL start reagent (APTT-SP CaCl 2) was added, and the solidification time was measured.
Chromogenic method에 의한 활성 측정은 CHROMOGENIX(社)에서 판매하고 있는 chromogenic assay kit를 endpoint 방법으로 사용하였고, 제조사가 제공한 시험방법을 다음과 같이 본 시험에 적합하게 변형하여 시험을 진행하였다. 구체적으로 시료를 1× 희석버퍼를 사용하여 standard range (0.25-1 IU/mL)에 들어오도록 희석한다. 표준품(calibration plasma)을 0, 0.25, 0.5, 1 IU/mL 농도가 되게 1x 희석버퍼를 이용하여 준비하였다. 이렇게 준비한 시료와 표준품 10μL를 1x 희석버퍼 790μL로 희석한 후, 96 well plate에 50μL씩 분주하고, 37℃에서 5분간 정치하였다. Automatic dispenser pipette을 사용하여 factor reagent를 50μL 씩 각 well에 분주한 후 2분간 37℃에서 2분간 반응하였다. 50μL substrate를 각 well에 분주한 후 2분간 37℃에서 2분간 정치하여 발색시켰다. 발색된 시료에 50mL 2% citric acid를 넣어주어 반응을 정지하였다. 405nm 파장에서 흡광도를 측정하여 선형 calibration curve를 그린 후, 샘플의 흡광도를 calibration curve에 대입하여 시료의 활성을 측정하였다. Chromogenic method was used to measure the activity of chromogenic assay kit sold by CHROMOGENIX as an endpoint method, and the test method provided by the manufacturer was modified to suit the present test as follows. Specifically, dilute the sample to enter the standard range (0.25-1 IU / mL) using a 1 × dilution buffer. Calibration plasma was prepared using a 1 × dilution buffer to a concentration of 0, 0.25, 0.5, 1 IU / mL. 10 μL of the prepared sample and 10 μL of the standard product were diluted with 790 μL of 1 × dilution buffer, and then 50 μL was dispensed into a 96 well plate and left at 37 ° C. for 5 minutes. 50 μL of the reagent reagent was dispensed into each well by using an automatic dispenser pipette, followed by reaction at 37 ° C. for 2 minutes. 50 μL substrates were dispensed into each well and allowed to develop for 2 minutes at 37 ° C. for 2 minutes. 50mL 2% citric acid was added to the developed sample to stop the reaction. After measuring the absorbance at 405nm wavelength to draw a linear calibration curve, the absorbance of the sample was substituted into the calibration curve to measure the activity of the sample.
측정된 결과는 표 2에 기재되었다. The measured results are listed in Table 2.
[표 2] 발현된 재조합 scFVIII의 활성 측정 결과Table 2 Results of Activity of Expressed Recombinant scFVIII
Figure PCTKR2017006633-appb-I000002
Figure PCTKR2017006633-appb-I000002
발현결과, 배양액 중 scFVIII 발현량은 B 영역의 길이가 증가함에 따라 증가하여 B 영역의 길이가 아미노산 198개 이상인 scF4, scF5, scF6, scF7 에서는 발현량이 현저히 증가됨을 확인하였다. 또한 B 영역의 길이가 아미노산 92개 이상인 scF3, scF4, scF5, scF6, scF7 에서는 비교 기준으로 사용한 재조합 형태의 전장의 FVIII (Advate®, Baxalta 제품)과 유사 수준의 비활성을 보였고 APTT/CS 비율에 있어서도 기준으로 사용한 Advate®와 유사하였다. 반면 scFVIII의 B 영역 길이가 상기 실시예의 scF1, scF2, scF3 같이 128개 아미노산 단백서열 길이보다 짧은 경우 발현량이 현저히 낮았으며 B 영역 길이가 상기 실시예의 scF1, scF2 같이 84개 아미노산 단백서열 길이보다 짧은 경우 비활성이 비교 기준으로 사용한 전장의 FVIII인 Advate® 보다 현저히 낮았다.As a result, the expression of scFVIII in the culture medium increased with the increase in the length of the B region, and it was confirmed that the expression level was significantly increased in scF4, scF5, scF6, scF7 having a length of the B region of 198 or more amino acids. In addition, scF3, scF4, scF5, scF6, and scF7, which have a length of 92 or more amino acids, showed similar levels of inactivation to the recombinant full-length FVIII (Advate®, manufactured by Baxalta) and used as a reference. Similar to Advate® used as reference. On the other hand, when the length of the B region of scFVIII is shorter than the length of 128 amino acid protein sequences such as scF1, scF2, and scF3 in the above example, the expression level was significantly lower, and the length of the B region was shorter than the length of 84 amino acid protein sequences such as scF1, scF2 of the Example. Inactivity was significantly lower than Advate®, the full-length FVIII used as a reference.
단쇄 FVIII은 활성화 단계에서 중쇄와 경쇄를 연결하는 B 영역을 포함하는 부위에서 트롬빈 절단에 의해 활성화된다. 중쇄와 경쇄의 연결 부위인 B 영역의 길이가 짧을 경우 짧은 연결체 길이에 의한 구조적 제한으로 단쇄 FVIII이 트롬빈에 의해 활성형 FVIII으로 전환이 정상적이지 않아 활성에 영향을 줄 것으로 판단된다. 그러나 이쇄 FVIII의 경우는 이와 같은 구조적 제한이 없어 B 영역의 길이에 상관없이 트롬빈에 의한 활성화가 원활할 것으로 판단되며 이는 기존의 B 영역이 완전 제거된 이쇄 FVIII의 활성이 B 영역을 포함하는 전장의 FVIII의 활성과 유사하다는 것으로부터 알 수 있다. 본원에서는 예기치 않은 노력을 통하여 단쇄 FVIII의 B 영역의 길이와 서열을, 트롬빈 활성화에 영향을 주지 않도록 선정하여 중쇄와 경쇄 사이의 트롬빈 활성화 부위(1689-1690 및 740-741; 서열번호 1의 서열 기준)가 천연형(즉 야생형)의 FVIII와 유사하게 활성화되어 비활성이, 비교로 사용한 전장의 FVIII외 유사한 단쇄 FVIII을 수득하였다.Short-chain FVIII is activated by thrombin cleavage at the site comprising the B region connecting the heavy and light chains in the activation step. If the length of the B region, which is the linking site between the heavy and light chains, is short, the structural limitation by the short linker length may affect the activity due to the abnormal conversion of short-chain FVIII to thrombin-activated FVIII. However, in the case of the double-chain FVIII, there is no structural limitation, and thus activation of thrombin regardless of the length of the B region is expected to be smooth. It can be seen from the similarity to the activity of FVIII. Through unexpected efforts herein, the length and sequence of the B region of the single-chain FVIII are selected so as not to affect thrombin activation, so that the thrombin activation sites (1689-1690 and 740-741; ) Was activated similar to the native (ie wild-type) FVIII, yielding similar short-chain FVIII in addition to the full-length FVIII used in comparison.
실시예 1-3. 구축된 scFVIII 발현 패턴 분석Example 1-3. Constructed scFVIII Expression Pattern Analysis
본원에 구축된 단쇄 FVIII의 배양에서의 안정성은 웨스턴블랏 분석을 통해 확인하였다. Stability in culture of the single chain FVIII constructed herein was confirmed by Western blot analysis.
이를 위해 배양액과 LDS sample buffer를 각각 3:1의 비율로 혼합하여 4-12% bis-tris(BT) 젤에 혼합한 시료 30μL를 로딩한 후 150 volt로 약 1시간 동안 러닝하였다. 러닝이 완료된 젤을 Nitrocellulose(NC) 막에 전달한 후, blocking 버퍼(Thermo Scientific)에 한 시간 정치하였다. 이어 FVIII의 heavy chain (GMA-012, Green Mountain Antibodies) 및 light chain (ab41188, Abcam)을 인식하는 1차 항체를 TBST 버퍼에 희석하여 제조한 후, blocking이 완료된 NC 막에서 넣어 주고 1시간 반응을 수행하였다. 이어 TBST 버퍼를 사용하여 5분 간격으로 3회 세척 후, TBST 버퍼로 희석된 2차 항체(Goat anti-mouse IgG-HRP conjugate)를 NC membrane와 10분간 반응시켰다. 이후 TBST 버퍼를 사용하여 5분 간격으로 5회 세척하였다. 이어 ECL Prime Western Blotting Detection Reagent을 뿌려주어 NC 막을 발색시킨 후, 암실에서 Hyperfilm ECL (GE healthcare)에 막을 현상하였다. 결과는 도 3에 기재된 바와 같이, 단쇄 형태로 제작된 FVIII은 모두 단쇄 형태로 발현되는 것을 확인하였다. 또한 배양액으로 분비된 단쇄 FVIII은 B 영역의 길이가 짧을수록 배양액에서 분해가 덜하여 더 안정한 것으로 나타났다. 상기 실시예에서와 같이 B 영역의 길이가 비교적 짧은 scF1, scF2, scF3의 안정성은 scF4, scF5, scF6, scF7 보다 우수하나 상대적으로 발현량이 낮거나 또는 비활성이 낮았다. To this end, the culture solution and LDS sample buffer were mixed at a ratio of 3: 1, respectively, and loaded with 30μL of the sample mixed in 4-12% bis-tris (BT) gel, and then run at 150 volt for about 1 hour. After running the gel was transferred to Nitrocellulose (NC) membrane, it was allowed to stand for one hour in blocking buffer (Thermo Scientific). Subsequently, primary antibodies that recognize heavy chains (GMA-012, Green Mountain Antibodies) and light chains (ab41188, Abcam) of FVIII were prepared by diluting them in TBST buffer, and then placed in a blocked NC membrane and reacted for 1 hour. Was performed. After washing three times at 5 minute intervals using TBST buffer, a secondary antibody (Goat anti-mouse IgG-HRP conjugate) diluted with TBST buffer was reacted with the NC membrane for 10 minutes. After washing 5 times at 5 minute intervals using the TBST buffer. Subsequently, the NC membrane was developed by spraying ECL Prime Western Blotting Detection Reagent, and then developed on Hyperfilm ECL (GE healthcare) in the dark. As a result, as shown in Figure 3, it was confirmed that all of the FVIII produced in the short-chain form is expressed in the short-chain form. In addition, the short-chain FVIII secreted into the culture medium was more stable because the shorter the length of the B region is less degradation in the culture medium. As in the above examples, the stability of scF1, scF2, and scF3 having a relatively short B region was superior to scF4, scF5, scF6, and scF7, but the expression level was relatively low or inactive.
본원에서는 천연형의 이쇄 FVIII으로부터 유래한 다양한 길이의 B 영역을 갖는 단쇄 FVIII을 제작하고 그 특성을 분석하였으며 B 영역의 길이가 발현량, 비활성, 안정성에 미치는 영향을 평가하여 천연형의 FVIII과 활성 특성과 유사하고 발현량이 높은 재조합 단쇄 FVIII을 제작할 수 있었다. In the present application, short-chain FVIII having various lengths of B region derived from natural type double-chain FVIII was prepared and characterized, and the effect of length of B region on the expression level, inactivation, and stability was evaluated to evaluate the effect of FVIII and activity of natural type. Recombinant single-chain FVIII with similar expression characteristics and high expression level could be produced.
실시예 2. scFVIII의 페길화Example 2. PEGylation of scFVIII
페길화 위치 선정Pegylation Location
B 영역의 일부를 포함하는 단쇄 FVIII에서 B 영역의 서열과 길이는 FVIII 활성 특성에 영향을 주어 재조합 혈액응고 FVIII의 활성과 특성에 영향을 줄 수 있기 때문에, 본 실시예에서는 천연형 이쇄 FVIII의 활성 특성을 보존할 수 있는 길이와 서열의 B 영역으로 구성된 실시예 1에서 제작된 scFVIII을 기본으로 체내 지속성을 부여하기 위하여 페길화 위치를 선정하였다. In the present embodiment, since the sequence and length of the B region in the single-chain FVIII including a part of the B region may affect the FVIII activity characteristics, the activity and properties of the recombinant blood coagulation FVIII may be affected. The PEGylation site was selected to give persistence in the body based on the scFVIII produced in Example 1, which is composed of the B region of the sequence and the length to preserve the properties.
B 영역내 페길화 반응 효율의 최적화를 위하여 B 영역의 여러 위치에서 페길화를 진행하였다. 페길화는 단백질의 표면 잔기를 대상으로 하기 때문에 페길화 위치를 선정하기 위해서는 B 영역의 3차원 구조가 필요하나, B 영역은 그 구조가 알려져 있지 않아 페길화 위치를 선정하는데 어려움 있었다. 본원에서 선정한 B 영역의 페길화 위치는 B 영역의 당쇄 주위를 선택적으로 페길화 하였다. 즉 당쇄는 단백질 구조의 밖에 위치하여 B 영역의 당쇄 주위 잔기는 항상 밖으로 향할 확률이 높아 페길화에 유리할 것으로 판단되었다. 그러나 당쇄에 밀접할수록 잔기는 당쇄에 의한 입체적 방해(steric hindrance)에 의해 PEG의 접근이 방해받아 페길화가 어려울 수 있는 문제점이 있었다. 본원에서는 예기치 않은 노력을 통해 당쇄로부터 적정 거리에 위치하여 B 영역의 단백 구조에서 밖으로 향하는 동시에 당쇄에 의해 steric hindrance를 받지 않아 페길화 효율이 보장되는 당쇄 근처의 잔기를 선정하였다.In order to optimize the PEGylation reaction efficiency in the B region, PEGylation was performed at various positions in the B region. Because PEGylation targets the surface residues of proteins, the three-dimensional structure of the B region is required to select the PEGylation position. However, since the structure is unknown, the PEGylation position was difficult to select. The PEGylation position of the B region selected herein selectively PEGylated around the sugar chain of the B region. In other words, the sugar chain is located outside the protein structure, and residues around the sugar chain of the B region are always likely to go out, and thus it may be advantageous for pegylation. However, the closer to the sugar chains, the more difficult the PEGylation of the residues may be due to the PEG's access is hampered by steric hindrance by the sugar chains. Through unexpected efforts, we selected residues near the sugar chain that are located at the proper distance from the sugar chain and are directed out of the protein structure of the B region and do not receive steric hindrance by the sugar chain, thereby ensuring PEGylation efficiency.
본 실시예에서는 표 3 및 도 4에 기재된 바와 같이 실시예 1에서 구축된 G4를 기준으로 B 영역의 당쇄 사슬 근처, 즉, N-당쇄 기준으로 +6, -6 잔기 사이의 다양한 위치에 시스테인이 도입된 재조합 scFVIII 변이체를 제작하고 도입된 시스테인에 선택적으로 페길화를 진행하였다. 이러한 페길화는 G4, single-chain FVIII를 대상으로 설명하였으나 동일한 논리로 본원에서 구축된 당쇄를 포함하는 다른 scFVIII의 페길화에도 적용될 수 있다. In this example, cysteine is located at various positions near the sugar chain of the B region based on G4 constructed in Example 1 as shown in Table 3 and FIG. 4, that is, between +6 and -6 residues based on the N-sugar chain. The introduced recombinant scFVIII variant was constructed and subjected to PEGylation selectively to the introduced cysteine. This PEGylation has been described for G4, single-chain FVIII, but can also be applied to the PEGylation of other scFVIII containing sugar chains constructed herein with the same logic.
실시예 2-1. 특정 부위가 시스테인으로 치환된 scFVIII 발현 벡터 제작Example 2-1. Construction of scFVIII Expression Vectors with Specific Sites Replaced with Cysteine
시스테인 치환 부위 및 FVIII heavy chain의 A2 영역 내에 존재하는 BamHI site (GGATCC)부터 light chain과 PacI 효소절단 부위가 포함되도록 GeneArt 사를 통해 유전자를 합성한 후 실시예 1-1에 제작된 발현벡터 pcDSW-scFVIII G4에 효소 BamHI/PacI을 이용하여 기존 FVIII의 해당 부위를 제거한 후 합성된 유전자를 BamHI/PacI site에 클로닝 하여 cysteine이 치환된 pcDSW-scFVIII G4 (B1, B2, B3, B4, B5, B6, B7, A3-1 또는 4L) 발현 벡터를 구축하였고, 발현 벡터 구축의 정확성은 서열분석을 통해 확인하였다. 그 외에 cysteine이 도입된 scFVIII G4 (A2-1, A2-2 또는 A2-3)은 cysteine 치환 부위를 포함하는 FVIII heavy chain의 A1 영역에 존재하는 AsisI 효소절단 부위부터 A2 영역 내에 존재하는 KpnI site (GGTACC)까지 GeneArt 사를 통해 유전자를 합성한 후 실시예 1-1에서 제작된 발현벡터 pcDSW-scFVIII G4에 효소 AsisI/KpnI을 이용하여 기존 FVIII의 해당 부위를 제거한 후 합성된 유전자를 AsisI/KpnI 부위에 클로닝 하여 cysteine이 치환된 pcDSW-scFVIII G4 (A2-1, A2-2 또는 A2-3)발현 벡터를 구축하였고, 발현 벡터 구축의 정확성은 서열분석을 통해 확인하였다. An expression vector prepared in Example 1-1 after synthesizing a gene through GeneArt to include a cysteine substitution site and a BamH I site (GGATCC) present in the A2 region of the FVIII heavy chain, and a light chain and a Pac I enzyme cleavage site. pcDSW-scFVIII G4 was removed using the enzyme BamH I / Pac I, and the cloned gene was cloned into BamH I / Pac I site to clone the synthesized pcDSW-scFVIII G4 (B1, B2, B3, B4, B5, B6, B7, A3-1 or 4L) expression vectors were constructed and the accuracy of expression vector construction was confirmed by sequencing. In addition the cysteine is introduced scFVIII G4 (A2-1, A2-2, or A2-3) are present in the Kpn I Asis I enzyme cleavage site from the area A2 present in the A1 region of the FVIII heavy chain comprising a cysteine substitution site site (GGTACC) by using the GeneArt Inc. enzymes Asis I / Kpn I in the expression vector pcDSW-scFVIII G4 prepared in example 1-1 was synthesized by the gene synthesis the gene, remove the corresponding parts of the conventional FVIII Cloning the Asis I / Kpn I site to construct a cysteine-substituted pcDSW-scFVIII G4 (A2-1, A2-2 or A2-3) expression vector, the accuracy of expression vector construction was confirmed by sequencing.
상기 구축된 cysteine이 도입된 mscFVIII 발현 벡터를 이용하여 실시예 1-2와 동일하게 일시 발현(transient expression)을 실시하였다. 각각의 시스틴 도입 FVIII의 발현량은 하기 표 3에 기재되어 있다. 표 3에서와 같이 cysteine이 B 영역의 glycan 사슬 주위에 도입된 scFVIII 변이체는 정상적으로 배양액에 발현되는 것으로 나타났다. 발현량은 cysteine 도입 이전의 scFVIII 보다 다소 낮은 수준이었으며 이는 scFVIII에 도입된 cysteine이 발현 과정 및 배양액 내에서 scFVIII의 안정성에 영향을 주기 때문인 것으로 생각된다.Transient expression was performed in the same manner as in Example 1-2 using the mscFVIII expression vector into which the constructed cysteine was introduced. The amount of expression of each cystine introduced FVIII is shown in Table 3 below. As shown in Table 3, the scFVIII variant in which cysteine was introduced around the glycan chain in the B region was normally expressed in the culture medium. The expression level was somewhat lower than scFVIII prior to cysteine introduction, which is thought to be because cysteine introduced into scFVIII affects the expression process and stability of scFVIII in culture.
실시예 2-2. 페길화 Example 2-2. Peg
페길화 위치를 스크리닝하기 위해 실시예 2-1에서와 같이 Cysteine이 도입된 scFVIII 변이체의 페길화는 다음과 같이 수행되었다. 세포 배양액 50mL을 5kDa MWCO (분자량 cutoff)로 20배 농축한 후, 최종 1M NaCl 되게 5M NaCl conc solution 첨가. 평형 버퍼 (20mM Histidine, 500mM NaCl, 0.1%(w/v) Tween®80, pH 7.0)로 평형화된 0.1mL 부피의 V8 select resin (GE 사)과 농축 시료를 충분히 함께 교반한 후 평형 버퍼로 세척 후 용출 버퍼 (20mM Histidine, 500mM NaCl, 0.1%(w/v) Tween®80, pH 7.0, 50%(v/v) propylene glycol)로 용출하였다. 용출된 시료는 5kDa MWCO 막을 사용하여 pegylation 버퍼 (20mM Histidine, 500mM NaCl, 0.1%(w/v) Tween®80, pH 7.0, 5%(w/v) sucrose)로 교환한 후 TCEP를 최종 0.2mM 처리하여 1시간 동안 4도에서 반응하였다. 이어 반응물의 TCEP를 제거하기 위해 페길화 버퍼 (20mM Histidine, 500mM NaCl, 0.1%(w/v) Tween®80, pH 7.0, 5%(w/v) sucrose)로 탈염 후, maleimide PEG 40kDa (NOF 사)를 1:20 몰비 (scFVIII : maleimide PEG 40kDa)로 첨가하여 1시간 동안 4도 조건에서 반응하였다.PEGylation of the scFVIII variant into which Cysteine was introduced as in Example 2-1 to screen for PEGylation sites was performed as follows. Concentrate 50 mL of the cell culture with 5 kDa MWCO (molecular weight cutoff) and add 5 M NaCl conc solution to the final 1 M NaCl. 0.1 mL volume of V8 select resin (GE) equilibrated with equilibration buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w / v) Tween®80, pH 7.0) and the concentrated sample were thoroughly stirred together and washed with equilibration buffer After elution with elution buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w / v) Tween®80, pH 7.0, 50% (v / v) propylene glycol). The eluted samples were exchanged with pegylation buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w / v) Tween®80, pH 7.0, 5% (w / v) sucrose) using a 5kDa MWCO membrane, followed by a final 0.2 mM of TCEP. Treated and reacted at 4 degrees for 1 hour. Then desalted with PEGylation buffer (20 mM Histidine, 500 mM NaCl, 0.1% (w / v) Tween®80, pH 7.0, 5% (w / v) sucrose) to remove TCEP of the reaction, followed by maleimide PEG 40kDa (NOF G) was added in a 1:20 molar ratio (scFVIII: maleimide PEG 40kDa) and reacted at 4 ° C. for 1 hour.
Cysteine이 도입된 scFVIII 변이체는 세포 배양을 통한 발현 시 도입된 cysteine이 cysteine 이나 glutathione에 의해 disulfide bond로 masking 되므로 페길화를 위해서는 masking 하고 있는 cysteine과 glutathione을 제거해야 한다. 본원에서는 도입된 cysteine의 자유 티올기를 masking 하고 있는 cysteine 이나 glutathione을 이탈시키기 위하여 TCEP(tris(2-carboxyethyl)phosphine)를 처리 후 탈염 컬럼(PD10)을 통하여 TCEP를 제거한 후 cysteine에 특이적인 maleimide-PEG (40kDa)를 처리하여 페길화 하였다.Cysteine-introduced scFVIII variants require cysteine and glutathione to be masked for pegylation because cysteine introduced during cell culture is masked as a disulfide bond by cysteine or glutathione. Herein, to remove cysteine or glutathione masking the free thiol group of cysteine introduced, TCEP (tris (2-carboxyethyl) phosphine) was treated, followed by removal of TCEP through desalination column (PD10), followed by maleimide-PEG specific for cysteine. (40 kDa) was treated to pegylated.
페길화 정도를 분석하기 위하여 페길화 반응물에 대한 SDS PAGE를 수행하였다. SDS PAGE was performed on the PEGylation reaction to analyze the degree of PEGylation.
결과는 도 6에 기재되어 있다. Cysteine이 도입된 G4scFVIII 변이체는 배양액 내에 활성 형태로 발현되었고 페길화가 용이하게 진행됨을 확인하였다. Cysteine 도입 위치에 따라 발현량과 페길화율에 차이가 있었으며 이는 도입된 cysteine 위치에 따라 FVIII의 안정성과 페길화 반응에 차이가 있기 때문인 것으로 생각된다. The results are described in FIG. 6. Cysteine-introduced G4scFVIII variant was expressed in the active form in the culture medium and it was confirmed that PEGylation proceeds easily. The expression level and PEGylation rate were different depending on the position of cysteine introduction, which may be due to the difference in stability and PEGylation of FVIII according to the position of cysteine introduced.
[표 3] scFVIII 접합부위가 cys로 치환된 mutant 발현량 및 페길화 효율Table 3 Expression of mutant and PEGylation efficiency of cys substituted scFVIII junction
Figure PCTKR2017006633-appb-I000003
Figure PCTKR2017006633-appb-I000003
*: 상기 표 3의 잔기는 서열번호 1의 서열을 기준으로 함*: The residues in Table 3 are based on the sequence of SEQ ID NO: 1
실시예 3. 페길화 scFVIII 생산 Example 3. Pegylated scFVIII Production
본 실시예에서는 실시예 2에서 선정된 위치에 페길화된 scFVIII를 다음과 같이 생산하였다. 또한 페길화 scFVIII을 생산하고 체내 지속형 혈액 응고 FVIII으로서의 활성과 페길화가 혈액 응고 FVIII의 활성에 미치는 영향을 평가하였다. G4, scFVIII의 B 영역 부위의 782번째 isoleucine이 cysteine으로 치환된 scFVIII인 B3의 위치 특이적 페길화 접합물을 생산 후 CS와 OS (APTT) 방법으로 활성을 측정하였고 이쇄 형태의 재조합 혈액 응고 FVIII인 Advate®와 비교하였다. In this example, pegylated scFVIII was produced at the position selected in Example 2 as follows. We also produced PEGylated scFVIII and evaluated the activity as sustained blood coagulation FVIII in the body and the effect of PEGylation on the activity of blood coagulation FVIII. The position-specific PEGylated conjugate of B3, scFVIII substituted with cysteine by G4, scFVIII's B region region, was measured for activity by CS and OS (APTT) method, and was a double-chain recombinant blood coagulation FVIII. Compared with Advate®.
실시예 3-1. 세포 배양Example 3-1. Cell culture
실시예 2-1에서 구축된 cysteine이 도입된 scFVIII G4 (B3)과 scFVIII G4 (A2_1)를 각각의 세포에서 발현하고 배양하였다. 구체적으로 우선 발현을 위해 실시예 2-1에서 구축된 발현 벡터를 Expi293F™ 세포에 Expi293F™ Expression System Kit (Thermofisher 사, Catalog Number A14635)을 제조자의 방법대로 사용하여 전달 이입하였다. 요약하면, 형질전환시 세포 수와 세포 생존율(viability)을 측정하여 세포 생존율이 95% 이상일 경우 형질전환을 진행하였다. 125mL flask에 7.5 X 107 cell 되도록 Expi293 배양배지를 첨가하여 25.5mL로 맞춰주었다. 실시예 2-2에서 구축된 발현 벡터 30μg를 Opti-MEM을 이용하여 총 볼륨이 1500μL이 되도록 섞어주었다. 80μL transfection reagent를 Opti-MEM을 이용하여 총 볼륨이 1500μL이 되도록 섞어주고 상온에서 5분간 배양하였다. 5분 후 transfection reagent가 들어있는 Opti-MEM을 DNA가 들어있는 Opti- MEM에 넣어주고 부드럽게 섞어주었다. 그리고 20~30분간 상온에서 반응시켜 주었다. 3mL의 DNA : transfection reagent 복합체를 미리 준비해 놓은 125mL flask Expi293F™ 세포에 (Total volume: 28.5mL)에 한 방울씩 떨어뜨려주고 37℃, 5% CO2 shacking incubator에서 125 rpm으로 배양하였다. 16~20시간 후에 Enhancer 1과 Enhancer 2를 각각 150μL, 1.5mL을 넣어주고 34℃, 5% CO2 교반인큐베이터에서 125 rpm으로 배양하였다. 배양 2일째 세포를 모두 원심분리하여 기존의 배양배지를 완전히 제거하고 30mL의 새로운 배양배지에 세포를 모두 풀어준 후 34℃, 5% CO2 shacking incubator에서 125 rpm으로 배양하였다. 도입 3일 차에 scFVIII를 발현하는 세포를 모두 원심 분리하여 배양액을 회수하였다. 배지 회수 후 남은 세포를 동일한 부피의 새로운 배양배지에 세포를 모두 풀어준 후 1일간 34℃에서 배양을 실시하였다. 본 과정을 배양 5일차까지 반복하여 3일, 4일, 5일 배양액을 회수하여 cysteine 도입된 scFVIII의 활성 분석 및 정제를 진행하였다.The cysteine constructed in Example 2-1 introduced scFVIII G4 (B3) and scFVIII G4 (A2_1) were expressed in each cell and cultured. Specifically, for expression, the expression vector constructed in Example 2-1 was transferred to Expi293F ™ cells using an Expi293F ™ Expression System Kit (Thermofisher, Catalog Number A14635) according to the manufacturer's method. In summary, the cell number and viability at the time of transformation were measured and transformation was performed when the cell viability was 95% or more. Into a 125mL flask was adjusted to 25.5mL by adding Expi293 culture medium to 7.5 X 10 7 cells. 30 μg of the expression vector constructed in Example 2-2 was mixed using Opti-MEM so that the total volume was 1500 μL. 80 μL transfection reagent was mixed to a total volume of 1500 μL using Opti-MEM and incubated for 5 minutes at room temperature. After 5 minutes, the Opti-MEM containing the transfection reagent was added to the Opti-MEM containing the DNA and mixed gently. And reacted at room temperature for 20-30 minutes. A 3 mL DNA: transfection reagent complex was previously dropped into a 125 mL flask Expi293F ™ cell (Total volume: 28.5 mL) and incubated at 125 rpm in a 37 ° C., 5% CO 2 shacking incubator. After 16-20 hours, 150 μL and 1.5 mL of Enhancer 1 and Enhancer 2 were added thereto, and the cells were incubated at 125 rpm in a 34 ° C., 5% CO 2 stirred incubator. On the second day of culture, all the cells were centrifuged to completely remove the existing culture medium. After releasing all the cells into a new 30mL culture medium, the cells were incubated at 125 rpm in 34 ° C and 5% CO 2 shacking incubator. On day 3, all cells expressing scFVIII were centrifuged to recover the culture solution. After releasing the medium, all the cells were released in a new culture medium of the same volume, and then cultured at 34 ° C. for 1 day. This procedure was repeated until the 5th day of culture to recover the culture medium for 3 days, 4 days, and 5 days, and the activity analysis and purification of cysteine-introduced scFVIII were performed.
실시예 3-2. 페길화 및 정제 Example 3-2. PEGylation and Purification
페길화 및 정제 공정은 크게 5단계로 구성되어 있으며, 실시예 3-1에서 배양한 3, 4, 5일차 배양액을 풀링(pooling)하여 정제를 진행하였다. The PEGylation and purification process is largely composed of five stages, and purification was performed by pooling the culture medium 3, 4, and 5 in Example 3-1.
첫 번째 단계에서는 배양액에서 FVIII을 분리정제 하는 공정으로 FVIII 정제용으로 GE에서 개발한 VIIISelect resin을 이용하였다. VIIISelect resin을 컬럼에 패킹한 후, 2% citric acid로 컬럼을 세척하였다. 평형 버퍼 (20mM Histidine, 5mM CaCl2, 1M NaCl, 0.02% Tween®80, pH 7.0)를 흘려주어 평형을 잡았다. 풀링한 배양액에 최종 1M NaCl 농도가 되도록 5M NaCl 버퍼를 넣어주고, pH를 7.0으로 적정한 후, 컬럼에 로딩하였다. 배양액 로딩 후, 평형 버퍼를 UV가 baseline으로 떨어질 때까지 흘려주어 평형을 잡았다. 용출 버퍼 (20mM Histidine, 5mM CaCl2, 0.9M Arginine, 45% propyleneglycol, 0.02% Tween®80, pH 6.5)를 흘려주어 scFVIII을 용출하였다.In the first step, the VIIISelect resin developed by GE for the purification of FVIII was used to isolate and purify FVIII from the culture. After packing VIIISelect resin into the column, the column was washed with 2% citric acid. The equilibration buffer (20 mM Histidine, 5 mM CaCl 2 , 1 M NaCl, 0.02% Tween® 80, pH 7.0) was run out to equilibrate. 5M NaCl buffer was added to the final culture solution to the final 1M NaCl concentration, the pH was adjusted to 7.0, and then loaded on the column. After incubation, the equilibration buffer was equilibrated by flowing until UV dropped to the baseline. Elution buffer (20 mM Histidine, 5 mM CaCl 2 , 0.9 M Arginine, 45% propyleneglycol, 0.02% Tween®80, pH 6.5) was run out to elute scFVIII.
이어 두 번째 단계에서는 첫 번째 단계의 용출액에 섞여 있는 propylene glycol을 빠른 속도로 제거해주는 공정으로 GE사의 SP fast flow resin을 이용하였다. SP fast flow resin을 컬럼에 패킹한 후, 세척 버퍼 (0.5M NaOH, 1M NaCl)를 흘려주어 컬럼을 세척한다. 평형 버퍼 (20mM Histidine, 5mM CaCl2, 0.02% Tween®80, pH 7.0)를 흘려주어 평형을 잡았다. VIIISelect 공정의 용출액을 평형 버퍼로 10배 희석한 후, pH를 7.0으로 적정하고, 컬럼에 로딩하였다. 로딩 후, 평형 버퍼를 UV가 baseline으로 떨어질 때까지 흘려주어 평형을 잡았다. 용출 버퍼 (20mM Histidine, 5mM CaCl2, 400mM NaCl, 0.02% Tween®80, pH 6.5)를 흘려주어 scFVIII을 빠르게 용출하였다.In the second step, GE's SP fast flow resin was used to rapidly remove propylene glycol in the eluate of the first step. After packing SP fast flow resin into the column, wash the column by flowing washing buffer (0.5M NaOH, 1M NaCl). Equilibration was performed by flushing equilibration buffer (20 mM Histidine, 5 mM CaCl 2 , 0.02% Tween® 80, pH 7.0). The eluate of the VIIISelect process was diluted 10-fold with equilibration buffer, then the pH was titrated to 7.0 and loaded onto the column. After loading, the equilibration buffer was equilibrated by flowing until UV dropped to the baseline. Elution buffer (20 mM Histidine, 5 mM CaCl 2 , 400 mM NaCl, 0.02% Tween® 80, pH 6.5) was run out to rapidly elute scFVIII.
세 번째 단계에서는 정제한 scFVIII에 PEG를 접합시켜 주는 공정이다. 정제된 scFVIII 용액에 최종농도가 0.1mM이 되도록 TCEP를 첨가한 후 4℃에서 1시간 정치시켜 삽입한 cysteine을 환원시켰다. 페길화 버퍼 (20mM Histidine, 5mM CaCl2, 200mM NaCl, 0.02% Tween®80, pH 7.0)로 평형이 잡힌 PD-10 컬럼(GE healthcare)을 사용하여 잔존 TCEP를 제거하였다. 환원된 FVIII 자체의 이황화 결합을 산화시켜주기 위하여 4℃에서 2시간 정치하였다. scFVIII G4 (B3)의 PEG 접합은 DMSO에 녹인 40kDa 크기의 branched 형태의 methoxy maleimide PEG 용액 (50mg/mL)을 단백질 당 PEG 비율을 1:20이 되도록 넣어준 후 4℃에서 12-16시간 정치하였다. scFVIII G4 (A2_1) PEG 접합은 60kDa 크기의 branched 형태의 methoxy maleimide PEG를 사용하여 동일하게 진행하였다.In the third step, PEG is conjugated to purified scFVIII. TCEP was added to the purified scFVIII solution so that the final concentration was 0.1 mM, and the cysteine was reduced by standing at 4 ° C for 1 hour. Residual TCEP was removed using a PD-10 column (GE healthcare) equilibrated with PEGylation buffer (20 mM Histidine, 5 mM CaCl 2 , 200 mM NaCl, 0.02% Tween® 80, pH 7.0). In order to oxidize the disulfide bond of the reduced FVIII itself, it was left at 4 ° C for 2 hours. PEG conjugation of scFVIII G4 (B3) was allowed to stand for 12-16 hours at 4 ° C after adding 40 kDa branched methoxy maleimide PEG solution (50mg / mL) dissolved in DMSO so that the PEG ratio per protein was 1:20. . The scFVIII G4 (A2_1) PEG conjugation was performed in the same manner using a 60 kDa branched methoxy maleimide PEG.
이어 네 번째 단계에서는 PEGylation 과정 중에 생긴 impurity인 PEG가 붙지 않은 scFVIII과 PEG가 2개 이상 붙은 scFVIII을 제거하는 공정으로 GE사의 SP fast flow resin을 이용하였다. SP fast flow resin을 컬럼에 패킹한 후, 세척 버퍼 (0.5M NaOH, 1M NaCl)를 흘려주어 컬럼을 세척하였다. 평형 버퍼 (20mM Histidine, 5mM CaCl2, 0.02% Tween®80, pH 7.0)를 흘려주어 평형을 잡았다. PEGylation 공정의 용출액을 평형 버퍼로 10배 희석한 후, pH를 7.0으로 적정하고, 컬럼에 로딩하였다. 로딩 후, 평형 버퍼를 UV가 베이스라인으로 떨어질 때까지 흘려주어 평형을 잡았다. 용출 버퍼 (20mM Histidine, 5mM CaCl2, 50-400mM NaCl, 0.02% Tween®80, pH 6.5)의 NaCl 농도를 단계별로 올려 주며, PEG-scFVIII을 선택적으로 용출하였다. In the fourth step, GE's SP fast flow resin was used to remove scFVIII without PEG and scFVIII with two or more PEGs, impurity generated during PEGylation. After packing SP fast flow resin in the column, the column was washed by flowing washing buffer (0.5M NaOH, 1M NaCl). Equilibration was performed by flushing equilibration buffer (20 mM Histidine, 5 mM CaCl 2 , 0.02% Tween® 80, pH 7.0). The eluate of the PEGylation process was diluted 10-fold with equilibration buffer, then the pH was titrated to 7.0 and loaded onto the column. After loading, the equilibration buffer was equilibrated by running until UV fell to the baseline. NaCl concentration in elution buffer (20 mM Histidine, 5 mM CaCl 2 , 50-400 mM NaCl, 0.02% Tween®80, pH 6.5) was stepped up, and PEG-scFVIII was eluted selectively.
마지막으로 다섯 번째 단계에서는 네 번째 단계에서 용출한 PEG-scFVIII을 농축하는 공정으로, 이를 위해 Millipore사의 amicon 30kDa를 이용하여 25 IU/mL 농도가 되게 농축을 하였다.Finally, in the fifth step, PEG-scFVIII eluted in the fourth step was concentrated. For this purpose, the concentration was 25 IU / mL using Millipore's amicon 30kDa.
실시예 3-3. 활성 측정Example 3-3. Active measurement
실시에 3-2에서 사용한 정제된 페길화 scFVIII (PEG-scFVIII-B3, PEG-scFVIII-A2-2)의 활성은 CS와 APTT 방법으로 측정하였다. CS 시험법은 실시예 1-2에 기재된 방법과 동일하게 수행하였고 APTT 시험법은 실시예 1-2에 기재된 방법을 변형하여 진행하였다. APTT 방법으로 페길화 FVIII의 활성 측정 시 활성제(activator)로 colloidal silica 계열의 APTT-SP를 사용하면 본래의 활성보다 낮은 활성 값이 분석되고 ellagic acid 계열의 synthAFax (IL) 사용하면 정상적인 활성이 측정된다고 알려져 있다 (Gu. J. M. et al., 2014. Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A. Haemophilia, 20, 593-600). 페길화 scFVIII 특성을 고려하여 APTT 시험 시 활성제로 synthAFax를 사용하였고 그 외 시험법은 실시예 1-2와 동일한 방법을 사용하였다. 결과는 표 4에 기재되어 있다. The activity of the purified PEGylated scFVIII (PEG-scFVIII-B3, PEG-scFVIII-A2-2) used in Example 3-2 was measured by CS and APTT methods. The CS test method was performed in the same manner as described in Example 1-2, and the APTT test method was performed by modifying the method described in Example 1-2. When the activity of PEGylated FVIII was measured by the APTT method, the colloidal silica-based APTT-SP was used as an activator, and the lower activity than the original activity was analyzed. When the ellagic acid synthAFax (IL) was used, the normal activity was measured. (Gu. JM et al., 2014. Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A. Haemophilia, 20, 593-600). In consideration of PEGylated scFVIII characteristics, synthAFax was used as an active agent in the APTT test, and the same method as in Example 1-2 was used. The results are shown in Table 4.
[표 4]TABLE 4
Figure PCTKR2017006633-appb-I000004
Figure PCTKR2017006633-appb-I000004
실험 결과, PEG-scFVIII-B3, PEG-scFVIII-A2-2은 비교물질로 사용한 전장의 B 영역을 포함하는 이쇄(two chain) FVIII인 Advate® 대비 APTT/CS 비율이(APTT/CS) 유사하였고 B 영역이 제거된 이쇄 FVIII인 Xyntha와 APTT/CS 비율이 상이함을 확인하였다. 이는 천연형의 FVIII의 활성이 CS와 APTT에서 거의 동일한 것을 고려할 때 본원에서 제작된 scFVIII 단백질은 단쇄화와 페길화에 의해 혈액 응고 FVIII의 활성 특성에 변화가 초래되지 않았음을 나타내는 것이다. Xyntha는 B-domain이 완전히 제거된 이쇄 재조합 FVIII으로 본 실시예에서 혈액응고 활성(APTT)이 CS에 비하여 감소하였으며 이는 혈액응고 활성이 B 영역의 완전 제거 시 감소된다는 기존의 보고와 상통한다. PEG-scFVIII-B3과 PEG-scFVIII-A2-2은 단쇄화 및 페길화 이후에도 혈액응고 활성이 유지되는 것을 고려할 때 본원에 따른 이 두 물질은 혈우병 치료제로서 장점을 갖는 것으로 판단된다. As a result, PEG-scFVIII-B3 and PEG-scFVIII-A2-2 showed similar APTT / CS ratio (APTT / CS) to Advate®, which is a two-chain FVIII containing full-length B region as a comparative material. It was confirmed that Xyntha, which is a double-chain FVIII from which the B region was removed, and the APTT / CS ratio were different. This indicates that the scFVIII protein produced herein did not result in a change in the activity properties of blood coagulation FVIII by short chain and PEGylation, considering that the activity of the native FVIII was almost the same in CS and APTT. Xyntha is a double-chain recombinant FVIII in which the B-domain is completely removed. In this example, coagulation activity (APTT) was reduced compared to CS, which is in line with previous reports that coagulation activity is reduced upon complete removal of the B region. Considering that PEG-scFVIII-B3 and PEG-scFVIII-A2-2 maintain blood coagulation activity even after short-chain and pegylation, these two substances according to the present application are considered to have advantages as therapeutic agents for hemophilia.
실시예 4. 동물 PK 시험Example 4. Animal PK Test
상기와 같이 페길화 scFVIII의 체내 반감기를 펜실바니아 대학에서 분양받아 번식하며 genotyping으로 확인된 11-12주령의 혈우병 A형 마우스 모델 (B6;129S4-F8tm1Kaz/J(FVIII-knock-out))에서 비교물질인 Advate®와 비교하였다. 시험 물질은 scFVIII인 G4, G4의 B 영역 부위의 782 isoleucine이 cysteine으로 치환된 후 40kDa branched PEG maleimide로 위치 특이적 페길화가 된 PEG40kDa-G4scFVIII-B3 (실시예 3 참조), G4의 A2 영역의 495번째 valine이 cysteine으로 치환된 후 60kDa branched PEG maleimide로 위치 특이적 페길화된 PEG60kDa-G4scFVIII-A2-2 그리고 이쇄의 재조합 FVIII인 Advate®이다.As described above, the half-life of pegylated scFVIII was distributed at the University of Pennsylvania, and it was compared and compared in 11-12-week-old hemophilia type A mouse model (B6; 129S4-F8tm1Kaz / J (FVIII-knock-out)) identified by genotyping. Compared with Advate®. The test substance was G4, scFVIII, PEG40kDa-G4scFVIII-B3 (see Example 3), which was site-specific PEGylated with 40kDa branched PEG maleimide after substitution of cysteine with 782 isoleucine at the B region of G4 (see Example 3), 495 of the A2 region of G4. The first valine is cy60e-substituted PEG60kDa-G4scFVIII-A2-2 with 60kDa branched PEG maleimide and Advate®, a recombinant FVIII of the double chain.
약물 약동학에 사용된 시험 동물은 혈우병 마우스(female)로 각 시료당 3마리의 혈우병 마우스에 125 IU/kg 수준으로 tail 정맥에 투여하였다. 투여 후 시간 별로(5min, 4hr, 8hr, 16hr, 24hr, 32hr, 40hr, 48hr, 56hr, 72hr) 혈액을 안와로부터 채취하여 항 응고제(3.2% sodium citrate)를 10% 처리한 후 원심 분리하여 혈장을 분리하였다. 혈장 내 존재하는 FVIII의 활성은 실시예 1-2에 기재된 바와 같이 CS assay 측정 시약을 사용하여 측정하였다. 시간별로 각 시료 군의 마우스로부터 채취한 혈장 내 FVIII 활성의 평균의 profile은 도 7과 같다.The test animals used for pharmacokinetics were hemophilia mice (females) administered to the tail vein at 125 IU / kg in three hemophilia mice per sample. Blood was collected from the orbit by time (5min, 4hr, 8hr, 16hr, 24hr, 32hr, 40hr, 48hr, 56hr, 72hr) after administration and treated with 10% anticoagulant (3.2% sodium citrate), followed by centrifugation. Separated. The activity of FVIII present in plasma was measured using CS assay measurement reagents as described in Examples 1-2. The profile of the average of FVIII activity in plasma collected from mice of each sample group by time is shown in FIG. 7.
PK parameter 분석은 혈장 내 FVIII CS 활성 측정값의 평균과 표준 편차를 WinNonlin software를 이용하여 NCA(Non Compartmental Analysis) 방법을 통해 산출하였으며 그 결과는 표 5와 같다. 약물의 반감기는 B3 ≒ A2-2 > G4 > Advate® 순으로 나타났다(표 5). Mean data를 기준으로 판단했을 때, 약물의 반감기는 Advate® 기준으로 B3는 약 1.9배, A2-2는 약 1.8배, G4는 약 1.2배 정도 증가 되었다 (표 5). The PK parameter analysis was performed using the NCA (Non Compartmental Analysis) method using the WinNonlin software to calculate the mean and standard deviation of plasma FVIII CS activity. The half-life of drugs was found in the order B3 ≒ A2-2> G4> Advate® (Table 5). Judging from the mean data, the half-life of drugs increased by 1.9 times for B3, 1.8 times for A2-2, and 1.2 times for G4 (Table 5).
[표 5] PK 결과Table 5 PK Results
Figure PCTKR2017006633-appb-I000005
Figure PCTKR2017006633-appb-I000005
실시예 5. 동물 PD 시험 (Long-term) Example 5. Animal PD Test (Long-term)
마우스에서 tail bleeding의 응고 효과를 다음과 같이 측정하였다. The coagulation effect of tail bleeding in mice was measured as follows.
실험개요는 도 8a에 기재된 바와 같다. 구체적으로 11-12주령의 수컷 C57BL/6 mouse(오리엔트 바이오 공급) 및 11-12주령의 펜실바니아 대학에서 분양받아 번식하며 genotyping으로 확인된 혈우병 A형 마우스 (B6;129S4-F8tm1Kaz/J(FVIII-knock-out), FVIII KO mice)를 시험에 사용하였다. C57BL/6 mouse는 7일 동안 순화과정을 거쳤다. 시험 당일, 15mL conical tube(1개/마리)에 saline을 14mL까지 채우고, 가열된 수조에 담가서 saline의 온도가 37℃가 되도록 맞추었다. 시험물질 투여 전 mouse의 체중을 측정하여 기록하였다. 엔토발(Pentobarbital Sodium)을 60mg/kg의 농도로 복강 내 투여하여 mouse를 마취하였다. 마우스 체중대비(마우스 체중은 19-25g 사이임) 시험 물질 투여량을 계산한 후, jugular vein으로 시험물질을 정맥 투여하였다. 투여 5분 후에 마우스 꼬리를 끝에서 4mm 절단한 후, 즉시 saline이 담긴 conical tube에 꼬리를 약 2cm 담그고, 30분간 유지시켜 혈액을 수집하였다. The experiment overview is as described in Figure 8a. Specifically, hemophilia type A mice (B6; 129S4-F8 tm1Kaz / J (FVIII-) identified by genotyping were bred by 11-12-week-old male C57BL / 6 mice (Orient Biosupply) and 11-12-week-old Pennsylvania University. knock-out), FVIII KO mice) were used for the test. C57BL / 6 mice were acclimated for 7 days. On the test day, saline was charged to 14 mL in a 15 mL conical tube (1 / horse) and soaked in a heated water bath to bring the saline temperature to 37 ° C. The weight of the mouse was measured and recorded before administration of the test substance. Mouse was anesthetized by intraperitoneal administration of Entobarbital Sodium at a concentration of 60 mg / kg. The test substance dose was calculated relative to the mouse body weight (mouse weight was between 19-25 g), and then the test substance was administered intravenously with jugular vein. After 5 minutes of administration, the mouse tail was cut 4 mm from the tip, and immediately after 2 cm of the tail was immersed in a conical tube containing saline, blood was collected for 30 minutes.
시험 물질은 실시예 3에서 제조된 PEG-scFVIII-B3로 Advate®와 비교하였다. 투여량(투여량 및 투여 부피) 그리고 각 시험 물질 군에 포함된 마우스의 배분은 아래 표 6과 같다.Test substances were compared to Advate® with PEG-scFVIII-B3 prepared in Example 3. Dose (dosage and dose volume) and distribution of mice in each test substance group are shown in Table 6 below.
[표 6]TABLE 6
Figure PCTKR2017006633-appb-I000006
Figure PCTKR2017006633-appb-I000006
각 마우스 별로 30분간 모은 혈액을 1500 x g에서 5분간 원심 분리하여 상층액을 제거한 후, 10mL pipet을 이용하여 conical tube에 3차 증류수를 10mL까지 넣은 후 vortex를 이용하여 혈액을 완전히 용혈시켰다. Blood loss 측정은 Hemoglobin assay kit(sigma, MAK115-1KT)에서 제공하는 매뉴얼에 따라 수행하였다. 요약하면, 1.5mL eppendorf tube에 3차 증류수 0.9mL과 용혈 샘플 0.1mL을 넣고 잘 섞어서 10배 희석한 후 96 well plate에 50μL의 3차 증류수 와 50μL의 calibrator를 넣고 (duplication) 그 위에 200μL의 3차 증류수를 넣었다. 각 마우스 용혈 샘플 50μL을 well에 넣고 (duplication), 그 위에 reagent 200μL을 넣었다. 넣은 물질들이 잘 섞일 수 있도록 96 well plate를 가볍게 두드린 후 Multimode plate reader를 이용하여 400nm에서 흡광도를 측정하였다. The blood collected for each mouse was centrifuged at 1500 x g for 5 minutes to remove the supernatant, and then the 10 mL pipet was used to add 3 mL of distilled water to the conical tube to 10 mL, and the blood was completely hemolyzed using vortex. Blood loss measurement was performed according to the manual provided in the Hemoglobin assay kit (sigma, MAK115-1KT). In summary, in a 1.5 mL eppendorf tube, add 0.9 mL of tertiary distilled water and 0.1 mL of hemolysis sample, mix well, dilute 10 times, add 50 μL of tertiary distilled water and 50 μL calibrator to a 96 well plate (duplication), and place 200 μL of 3 on top of it. Tea distilled water was added. 50 μL of each mouse hemolysis sample was placed in a well (duplication), and 200 μL of the reagent was placed thereon. After tapping the 96 well plate so that the materials were well mixed, the absorbance was measured at 400 nm using a Multimode plate reader.
흡광도 측정값은 GraphPad Prism ver 5.01의 One way ANOVA with Dunnett’s multiple comparison test를 사용하여 blood loss 값(Hb nmol)을 통계적으로 비교하였다. p-value가 0.05보다 클 경우, 통계적으로 유의한 차이가 아니라고 평가하고, p-value가 0.05보다 작을 경우, 통계적으로 유의한 차이라고 평가하였다. Absorbance measurements were statistically compared with blood loss values (Hb nmol) using One way ANOVA with Dunnett's multiple comparison test of GraphPad Prism ver 5.01. If the p-value is greater than 0.05, it is not statistically significant. If the p-value is less than 0.05, it is evaluated to be a statistically significant difference.
시험 결과 40kDaPEG-G4scFVIII-B3 및 Adate 50, 100, 200 IU/kg 투여군의 blood loss 값은 HA군의 blood loss 값과 비교 시, 모두 통계적으로 유의한 차이를 보였으며, HA mouse에 40kDaPEG-G4scFVIII-B3과 Advate®를 투여한 후, acute tail-clipping test를 진행한 결과 두 시험 물질 모두 농도 의존적으로 blood loss의 양을 감소시키는 것으로 나타났다 (도 8b 참조). As a result, the blood loss values of 40kDaPEG-G4scFVIII-B3 and Adate 50, 100, 200 IU / kg group were significantly different from those of HA group, and 40kDaPEG-G4scFVIII- in HA mouse. After administration of B3 and Advate®, the acute tail-clipping test showed that both test substances reduced the amount of blood loss in a concentration-dependent manner (see FIG. 8B).
정리하면 본원에서는 발현이 우수하고 천연 FVIII 활성을 최대한 보유하고 안정하여 배양액에서 분해가 방지되는 scFVIII을 선정하기 위하여 다양한 길이와 서열의 B 영역을 포함하는 scFVIII을 제작하고 발현한 후 활성을 측정하고, 안정성을 확인하는 과정을 통하여 혈우병 A형 치료제로서 유용한 특성을 보유한 scFVIII을 개발하였다. 또한 본원에서 개발한 scFVIII을 바탕으로 다양한 페길화 접합체를 제작하여 체내 반감기가 향상되어 치료제로서 유용성이 향상된 FVIII 페길화 접합체를 개발하였다. In summary, in the present application, in order to select scFVIII having excellent expression, retaining and maximally retaining natural FVIII activity and preventing degradation in culture, scFVIII including B regions of various lengths and sequences were prepared and expressed, and then the activity was measured. Through the process of confirming the stability, scFVIII was developed which has useful properties as a hemophilia type A therapeutic agent. In addition, various PEGylated conjugates were prepared based on the scFVIII developed in the present application, thereby improving FVIII PEGylated conjugates having improved usefulness as therapeutic agents.
이상에서 본원의 예시적인 실시예에 대하여 상세하게 설명하였지만 본원의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본원의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본원의 권리범위에 속하는 것이다.Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다. All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

Claims (23)

  1. 인간 혈액응고 8인자(Factor VIII)의 중쇄, 경쇄 및 일부가 결실된 B 영역 단편을 포함하며, 상기 일부가 결실된 B 영역 단편은 퓨린 단백질분해효소에 의한 절단부위를 포함하지 않도록 서열번호 1의 서열의 1648 및 1649 잔기를 포함하여 상기 잔기의 N-말단 및 C-말단 방향으로 각각 최소 5개의 아미노산이 결실되고, 4개 내지 6개의 당화 부위를 포함하도록 일부가 결실된 것인, 단쇄 혈액응고 8인자(Factor VIII). A heavy region, a light chain, and a portion of a B region fragment deleted from human coagulation factor 8 (Factor VIII), wherein the portion of the deleted B region fragment of SEQ ID NO: 1 does not include a cleavage site by purine protease. Short-chain blood clotting wherein at least 5 amino acids are deleted in the N-terminal and C-terminal directions of the residue, including 1648 and 1649 residues of the sequence, respectively, and partly deleted to contain 4 to 6 glycosylation sites Factor VIII.
  2. 제 1 항에 있어서, 상기 B 영역 단편은 야생형 B 영역 서열의 15% 내지 40%를 포함하는 것인, 단쇄 혈액응고 8인자. The factor of claim 1, wherein the B region fragment comprises 15% to 40% of the wild type B region sequence.
  3. 제 2 항에 있어서, The method of claim 2,
    상기 일부가 결실된 B 영역 단편은 서열번호 1의 서열을 기준으로 (i) 아미노산 잔기 741 내지 902 및 1654 내지 1689; (ii) 아미노산 잔기 741 내지 965 및 1654 내지 1689의 아미노산 서열; 또는 (iii) 아미노산 잔기 741 내지 902, 1637 내지 1642 및 1654 내지 1689;의 서열인, 단쇄 혈액응고 8인자. The B region fragment, wherein said portion is deleted, may be selected from (i) amino acid residues 741-902 and 1654-1689 based on the sequence of SEQ ID NO: 1; (ii) amino acid sequences of amino acid residues 741 to 965 and 1654 to 1689; Or (iii) the sequence 8 of amino acid residues 741 to 902, 1637 to 1642 and 1654 to 1689.
  4. 제 3 항에 있어서, The method of claim 3, wherein
    상기 단쇄 혈액응고 8인자는 서열번호 4, 서열번호 5 또는 서열번호 6으로 표시되는 서열 또는 이와 90% 이상 상동성이 있는 서열을 갖는 것인, 단쇄 혈액응고 8인자.The single-chain coagulation factor 8 is to have a sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or a sequence 90% or more homologous thereto, single-chain coagulation factor 8.
  5. 제 1 항 내지 제 4 항 중 어느 한 항에 있어서, The method according to any one of claims 1 to 4,
    상기 단쇄 혈액응고 8인자는 CS 또는 APTT 방법으로 측정한 비활성이 이쇄 혈액응고 8인자 비활성의 90% 이상인, 단쇄 혈액응고 8인자. The short-chain coagulation factor 8 is a short-chain coagulation factor 8, the inactivation measured by the CS or APTT method is 90% or more of the double-chain coagulation factor 8 inactivity.
  6. 제 1 항 내지 제 4 항 중 어느 한 항에 있어서, The method according to any one of claims 1 to 4,
    상기 혈액응고 8인자는 상기 A 영역 및/또는 B 영역 단편의 일부 아미노산 잔기에서 친수성 폴리머와 컨쥬게이션되고, 상기 컨쥬게이션되는 위치는 상기 B 영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 754, 781, 782, 788, 789, 825 및 897로 구성되는 군으로부터 선택되는 하나 이상의 위치이고; 상기 A-영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 491, 495, 498 및 1806으로 구성되는 군으로부터 선택되는 하나 이상의 위치이고, The coagulation factor 8 is conjugated with a hydrophilic polymer at some amino acid residues of the A region and / or B region fragment, and the conjugated position is based on the amino acid residues 754, 781 in SEQ ID NO: 1 in the B region. At least one position selected from the group consisting of 782, 788, 789, 825 and 897; At least one position selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1 in the A-region,
    상기 컨쥬게이션되는 위치의 잔기는 상기 친수성 폴리머와의 접합을 위해 시스테인으로 치환되는, 단쇄 혈액응고 8인자. Wherein the residue at the conjugated position is substituted with cysteine for conjugation with the hydrophilic polymer.
  7. 제 6 항에 있어서, The method of claim 6,
    상기 친수성 폴리머는 폴리에틸렌글리콜(Polyethylene glycol), 폴리에틸렌 옥사이드, 덱스트란 또는 폴리시알릭을 포함하고, 상기 PEG는 상기 위치에서 acryloyl, sulfone 또는 maleimide를 통하여 연결된 것인, 단쇄 혈액응고 8인자.Wherein said hydrophilic polymer comprises polyethylene glycol, polyethylene oxide, dextran or polysialic, and said PEG is linked via acryloyl, sulfone or maleimide at said position.
  8. 제 5 항에 있어서, The method of claim 5,
    상기 PEG는 평균 분자량이 20kDa 이상인 단쇄 혈액응고 8인자. The PEG has a short-chain coagulation factor 8 having an average molecular weight of 20 kDa or more.
  9. 제 1 항에 있어서, 상기 단쇄 혈액응고 8인자는 서열번호 2 내지 7 중 어느 하나의 아미노산 서열로 표시되는, 단쇄 혈액응고 8인자. The single-chain coagulation factor 8 of claim 1, wherein the single-chain coagulation factor 8 is represented by an amino acid sequence of any one of SEQ ID NOs: 2-7.
  10. 제 9 항에 있어서, 상기 단쇄 혈액응고 8인자는 서열번호 2 내지 7에서 제 6 항에 기재된 컨쥬게이션 부위가 시스테인으로 치환된 아미노산 서열을 갖는, 단쇄 혈액응고 8인자. The single-chain coagulation factor 8 according to claim 9, wherein the single-chain coagulation factor 8 has an amino acid sequence in which the conjugation site according to claim 6 in SEQ ID NOs: 2 to 7 is substituted with cysteine.
  11. 제 9 항 또는 제 10 항에 따른 단백질을 코딩하는 핵산분자. A nucleic acid molecule encoding a protein according to claim 9.
  12. 제 11 항에 있어서, 상기 제 9 항에 따른 핵산분자는 서열번호 10 내지 15 중 어느 하나이고, 상기 제 10 항에 따른 단백질을 코딩하는 핵산분자는 서열번호 17 내지 32 중 어느 하나인, 핵산분자. The nucleic acid molecule according to claim 11, wherein the nucleic acid molecule according to claim 9 is any one of SEQ ID NOs: 10 to 15, and the nucleic acid molecule encoding the protein according to claim 10 is any one of SEQ ID NOs: 17 to 32. .
  13. 제 11 항 또는 제 12 항에 따른 핵산분자를 포함하는 벡터. A vector comprising the nucleic acid molecule according to claim 11.
  14. 제 13 항에 따른 벡터를 포함하는 세포. A cell comprising the vector according to claim 13.
  15. 제 13 항에 따른 벡터를 진핵세포에 전달이입하는 단계; 또는 선택적으로 제 14 항에 따른 세포를 제공하는 단계; Transfecting the vector of claim 13 into a eukaryotic cell; Or optionally providing a cell according to claim 14;
    상기 세포를 배양액 중에서 배양을 통하여 도입된 시스테인이 자유 티올 기를 시스테인 또는 글루타티온에 의해 이황화(disulfide) 결합으로 마스킹 된 단쇄 FVIII형태로 발현시키는 단계; Expressing the cells in the culture medium in the form of single-chain FVIII masked with disulfide bonds by cysteine or glutathione-induced cysteine;
    상기 배양액으로부터 발현된 단쇄 FVIII을 수집하여 환원제로 처리하여 마스킹 된 시스테인이나 글루타티온을 이탈 시키는 단계; 및Collecting the short-chain FVIII expressed from the culture solution and treating it with a reducing agent to release masked cysteine or glutathione; And
    상기 처리된 배양액을 페길화완충액으로 처리하는 단계를 포함하는, 단쇄 혈액응고 8인자의 생산방법. Comprising the step of treating the treated culture solution with PEGylated buffer, the method of producing a short-chain blood coagulation factor 8.
  16. 인간 혈액응고 8인자(Factor VIII)의 중쇄, 경쇄 및 일부가 결실된 B 영역 단편을 포함하며, 상기 일부가 결실된 B 영역 단편은 퓨린 단백질분해효소에 의한 절단부위를 포함하지 않으며 최소 4개의 당화 부위를 포함하도록 그 일부가 결실된 것으로, Heavy chain, light chain and partly deleted B region fragment of human coagulation factor 8 (Factor VIII), which partly deleted B region fragment does not include cleavage site by purine protease and has at least 4 glycosylation Part of which is deleted to include the site,
    상기 일부가 결실된 B 영역 단편은 서열번호 1의 서열을 기준으로 (i) 아미노산 잔기 741 내지 902 및 1654 내지 1689; (ii) 아미노산 잔기 741 내지 965 및 1654 내지 1689의 아미노산 서열; 또는 (iii) 아미노산 잔기 741 내지 902, 1637 내지 1642 및 1654 내지 1689;의 서열인, 단쇄 혈액응고 8인자.The B region fragment, wherein said portion is deleted, may be selected from (i) amino acid residues 741-902 and 1654-1689 based on the sequence of SEQ ID NO: 1; (ii) amino acid sequences of amino acid residues 741 to 965 and 1654 to 1689; Or (iii) the sequence 8 of amino acid residues 741 to 902, 1637 to 1642 and 1654 to 1689.
  17. 인간 혈액응고 8인자(Factor VIII)의 중쇄, 경쇄 및 단백질분해효소에 의한 절단부위를 포함하지 않으며 최소 4개의 당화 부위를 포함하도록 일부가 결실된 B 영역 단편을 포함하는 단쇄 혈액응고 8인자로, As a single-chain coagulation factor 8 which includes a B region fragment which does not include the heavy, light and proteolytic cleavage sites of the human coagulation factor 8 (Factor VIII) and includes at least four glycosylation sites,
    상기 일부가 결실된 B 영역 단편은 서열번호 1의 서열을 기준으로 (i) 아미노산 잔기 741 내지 902 및 1654 내지 1689; (ii) 아미노산 잔기 741 내지 965 및 1654 내지 1689의 아미노산 서열; 또는 (iii) 아미노산 잔기 741 내지 902, 1637 내지 1642 및 1654 내지 1689; 의 서열이며, The B region fragment, wherein said portion is deleted, may be selected from (i) amino acid residues 741-902 and 1654-1689 based on the sequence of SEQ ID NO: 1; (ii) amino acid sequences of amino acid residues 741 to 965 and 1654 to 1689; Or (iii) amino acid residues 741-902, 1637-1642 and 1654-1689; Is a sequence of,
    상기 A 영역 및/또는 B 영역의 일부 잔기가 페길화되었으며, 상기 페길화된 잔기는 상기 B 영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 754, 781, 782, 788, 789, 825 및 897로 구성되는 군으로부터 선택되는 하나 이상의 위치이고; 상기 A-영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 491, 495, 498 및 1806으로 구성되는 군으로부터 선택되는 하나 이상의 위치인, 단쇄 혈액응고 8인자. Some residues of the A and / or B region have been pegylated, and the PEGylated residue is amino acid residues 754, 781, 782, 788, 789, 825 and 897 based on the sequence of SEQ ID NO: 1 in the B region. One or more positions selected from the group consisting of; Single chain hemagglutination factor 8 which is one or more positions selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1 in the A-region.
  18. 제 17 항에 있어서, The method of claim 17,
    상기 페길화가 되는 잔기는 시스테인으로 치환된 것인, 단쇄 혈액응고 8인자. The PEGylated residue is substituted with cysteine, short-chain blood coagulation factor 8.
  19. 인간 혈액응고 8인자(Factor VIII)의 중쇄, 경쇄 및 단백질분해효소에 의한 절단부위를 포함하지 않으며, 최소 4개의 당화 부위를 포함하도록 일부가 결실된 B 영역 단편을 포함하는 단쇄 혈액응고 8인자(Factor VIII)로, Single-chain coagulation factor 8, which contains a B region fragment which does not include the heavy, light and proteolytic cleavage sites of human coagulation factor 8 (Factor VIII) and which contains at least four glycosylation sites ( Factor VIII),
    상기 일부가 결실된 B 영역 단편은 서열번호 1의 서열을 기준으로 (i) 아미노산 잔기 741 내지 902 및 1654 내지 1689; (ii) 아미노산 잔기 741 내지 965 및 1654 내지 1689의 아미노산 서열; 또는 (iii) 아미노산 잔기 741 내지 902, 1637 내지 1642 및 1654 내지 1689;의 서열이며, The B region fragment, wherein said portion is deleted, may be selected from (i) amino acid residues 741-902 and 1654-1689 based on the sequence of SEQ ID NO: 1; (ii) amino acid sequences of amino acid residues 741 to 965 and 1654 to 1689; Or (iii) the amino acid residues 741-902, 1637-1642 and 1654-1689;
    상기 A 영역 및/또는 B 영역의 일부 잔기가 페길화되며, 상기 페길화된 잔기는 상기 B 영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 754, 781, 782, 788, 789, 825 및 897로 구성되는 군으로부터 선택되는 하나 이상의 위치이고; 상기 A-영역에서 서열번호 1의 서열을 기준으로 아미노산 잔기 491, 495, 498 및 1806으로 구성되는 군으로부터 선택되는 하나 이상의 위치인, 그리고 상기 페길화된 잔기는 시스테인으로 치환된 것인, 단쇄 혈액응고 8인자. Some residues of the A and / or B region are PEGylated, wherein the PEGylated residue is amino acid residues 754, 781, 782, 788, 789, 825 and 897 based on the sequence of SEQ ID NO: 1 in the B region. One or more positions selected from the group consisting of; Short-chain blood, wherein the PEGylated residue is at least one position selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO: 1 in the A-region; Coagulation factor 8.
  20. 제 1 항 내지 제 10 항 또는 제 16 항 내지 제 19 항 중 어느 한 항에 따른 유효한 양의 단쇄 혈액응고 8인자를 혈우병 치료가 필요한 대상체에게 처리하는 단계를 포함하는, 혈우병 치료 방법. 20. A method for treating hemophilia comprising treating an effective amount of the short-chain coagulation factor 8 according to any one of claims 1 to 10 or 16 to 19 to a subject in need of treatment for hemophilia.
  21. 제 1 항 내지 제 10 항 또는 제 16 항 내지 제 19 항 중 어느 한 항에 따른 유효한 양의 단쇄 혈액응고 8인자를 혈우병 치료가 필요한 대상체에게 처리하는 단계를 포함하는, 혈액 응고 방법. 20. A method of blood clotting comprising treating an effective amount of the short-chain clotting factor 8 according to any one of claims 1 to 10 or 16 to 19 to a subject in need of treatment for hemophilia.
  22. 제 1 항 내지 제 10 항 또는 제 16 항 내지 제 19 항 중 어느 한 항에 따른 단쇄 혈액응고 8인자 및 약학적으로 허용가능한 담체를 포함하는, 혈우병 치료용 조성물. 20. A composition for the treatment of hemophilia comprising the single chain coagulation factor 8 according to any one of claims 1 to 10 or 16 to 19 and a pharmaceutically acceptable carrier.
  23. 제 1 항 내지 제 10 항 또는 제 16 항 내지 제 19 항 중 어느 한 항에 따른 단쇄 혈액응고 8인자의 혈우병 치료용 용도. Use for the treatment of hemophilia of the short-chain coagulation factor 8 according to any one of claims 1 to 10 or 16 to 19.
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